http://www.cambridge.org/9780521855860

This page intentionally left blank

Dendritic Cell Interactions with Bacteria

Emerging evidence suggests that dendritic cells play a major role in the

orchestration of the immune response to bacteria. This book introduces the

reader to the complex world of dendritic cells and describes how the intimate

interplay between dendritic cells, bacteria and the environment dictates

either the induction of immunity or tolerance to the encountered micro-

organisms. It discusses how this can allow organisms to tolerate beneficial

bacteria and to react against pathogens, as well as the strategies pathogenic

bacteria have evolved to escape dendritic cell patrolling. Expert contributors

discuss everything from bacterial capture and recognition to their killing,

processing and the induction of adaptive immunity. Particular focus is on

the tissue context in which bacteria are handled by dendritic cells and on

possible defects therein, which may potentially lead to chronic infection or

inflammation. Graduate students and researchers will find this an invaluable

overview of current dendritic cell biology research.

MARIA RESCIGNO is the Director of the Immunobiology of Dendritic Cells

and Immunotherapy Research Unit at the European Institute of Oncology,

Milan, Italy.

Published titles

1 Bacterial Adhesion to Host Tissues. Edited by Michael Wilson 0521801079

2 Bacterial Evasion of Host Immune Responses. Edited by Brian Henderson

and Petra Oyston 0521801737

3 Dormancy in Microbial Diseases. Edited by Anthony Coates 0521809401

4 Susceptibility to Infectious Diseases. Edited by Richard Bellamy 0521815258

5 Bacterial Invasion of Host Cells. Edited by Richard Lamont 0521809541

6 Mammalian Host Defense Peptides. Edited by Deirdre Devine and Robert

Hancock 0521822203

7 Bacterial Protein Toxins. Edited by Alistair Lax 052182091X

8 The Dynamic Bacterial Genome. Edited by Peter Mullany 0521821576

9 Salmonella Infections. Edited by Pietro Mastroeni and Duncan Maskell

0521835046

10 The Influence of Cooperative Bacteria on Animal Host Biology. Edited by

Margaret McFall Ngai, Brian Henderson and Edward Ruby 0521834651

11 Bacterial Cell-to-Cell Communication. Edited by Donald R. Demuth and

Richard Lamont 0521846382

12 Phagocytosis of Bacteria and Bacterial Pathogenicity. Edited by Joel Ernst

and Olle Stendahl 0521845696

13 Bacterial-Epithelial Cell Cross-Talk: Molecular Mechanisms in Pathogenesis.

Edited by Beth A. McCormick 0521852447

Over the past decade, the rapid development of an array of techniques in the

fields of cellular and molecular biology has transformed whole areas of

research across the biological sciences. Microbiology has perhaps been

influenced most of all. Our understanding of microbial diversity and

evolutionary biology, and of how pathogenic bacteria and viruses interact

with their animal and plant hosts at the molecular level, for example, have

been revolutionized. Perhaps the most exciting recent advance in micro-

biology, a fusion of classical microbiology, microbial molecular biology and

eukaryotic cellular microbiology. Cellular microbiology is revealing how

pathogenic bacteria interact with host cells in what is turning out to be a

complex evolutionary battle of competing gene products. Molecular and

cellular biology are no longer discrete subject areas but vital tools and an

integrated part of current microbiological research. As part of this revolution

in molecular biology, the genomes of a growing number of pathogenic and

model bacteria have been fully sequenced, with immense implications for

our future understanding of microorganisms at the molecular level.

Advances in Molecular and Cellular Microbiology is a series edited by

researchers active in these exciting and rapidly expanding fields. Each

volume will focus on a particular aspect of cellular or molecular microbiology

and will provide an overview of the area, as well as examine current research.

This series will enable graduate students and researchers to keep up with the

rapidly diversifying literature in current microbiological research.

Series Editors

Professor Brian Henderson

University College, London

Professor Michael Wilson

University College, London

Professor Sir Anthony Coates

St George’s Hospital Medical School, London

Professor Michael Curtis

St Bartholomew’s and Royal London Hospital, London

MOLECU

LARAN

DCELLULARMICRO

BIOLOGY

AMCM

ADVANCESIN

Advances in Molecular and Cellular Microbiology 14

Dendritic Cell Interactionswith Bacteria

EDITED BY

MARIA RESCIGNOEuropean Institute of Oncology

CAMBRIDGE UNIVERSITY PRESS

Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo

Cambridge University PressThe Edinburgh Building, Cambridge CB2 8RU, UK

First published in print format

ISBN-13 978-0-521-85586-0

ISBN-13 978-0-511-29471-6

© Cambridge University Press 2007

2007

Information on this title: www.cambridge.org/9780521855860

This publication is in copyright. Subject to statutory exception and to the provision of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press.

ISBN-10 0-511-29471-9

ISBN-10 0-521-85586-1

Cambridge University Press has no responsibility for the persistence or accuracy of urls for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate.

Published in the United States of America by Cambridge University Press, New York

www.cambridge.org

hardback

eBook (EBL)

eBook (EBL)

hardback

Contents

Preface page ix

List of Abbreviations xiii

List of Contributors xvii

I Dendritic cells and their role in immunity 1

1 Subpopulations and differentiation of mouse dendritic cells 3

Carlos Ardavın

2 Toll-like receptor signaling 27

Osamu Takeuchi and Shizuo Akira

3 MHC class I and II pathways for presentation and

cross-presentation of bacterial antigens 51

Laurence Bougneres-Vermont and Pierre Guermonprez

II Dendritic cells and innate immune responsesto bacteria 79

4 Dendritic cell activation and uptake of bacteria in vivo 81

Maria Rescigno

5 Role of dendritic cells in the innate response to bacteria 99

Natalya V. Serbina and Eric G. Pamer

6 Interactions between natural killer and dendritic cells

during bacterial infections 119

Guido Ferlazzo

III Dendritic cells and adaptive immuneresponses to bacteria 139

7 Peculiar ability of dendritic cells to process and present

antigens from vacuolar pathogens: a lesson from Legionella 141

Sunny Shin, Catarina Nogueira and Craig R. Roy

8 Dendritic cells, macrophages and cross-presentation of

bacterial antigens: a lesson from Salmonella 159

Mary Jo Wick

IV Dendritic cells and immune evasionof bacteria in vivo 171

9 Pathogen-recognition receptors as targets for pathogens

to modulate immune function of antigen-presenting cells 173

Anneke Engering, Sandra J. van Vliet, Estella A. Koppel,

Teunis B.H. Geijtenbeek and Yvette van Kooyk

10 Suppression of immune responses by bacteria and their

products through dendritic cell modulation and regulatory

T cell induction 193

Miriam T. Brady, Peter McGuirk and Kingston H.G. Mills

11 Dendritic cells in the gut and their possible role in disease 223

Christoph Becker

Index 243

Colour plate section appears between pages 12 and 13

�

viii

CO

NT

EN

TS

Preface

Dendritic cells (DCs) comprise a family of professional antigen presenting

cells that are unique in their ability to activate T lymphocytes. Dendritic cells

patrol all the tissues at the interface with the external world, including skin

and mucosal surfaces, for the presence of invaders. The DC system is

characterized by a remarkable plasticity that allows the induction both of

immunity and tolerance toward the encountered antigens. This is achieved

through the combination of a number of different factors, including the

subsets of DCs, their activation state and environmental cells that can

regulate DC function. DCs are present in the periphery in an immature form

that is particularly apt at capturing antigens and at deciphering the messages

associated therein. After an activation stimulus that is delivered by some

antigens (including bacteria) or by inflammatory cytokines released during

inflammation, activated DCs acquire a migratory phenotype and reach

the draining lymph node. Here, DCs present the antigens captured in the

periphery and initiate T cell adaptive immune responses.

This book describes how the intimate interplay between dendritic cells,

bacteria and the environment dictates the induction of immunity or

tolerance to bacteria and how pathogenic bacteria have evolved strategies

to escape DC patrolling. The first section introduces the complexity of the DC

system describing the different subpopulations of DCs and their role in the

induction of immune responses. This is followed by the description of a class

of pathogen recognition receptors and their signaling pathways that are

fundamental in the activation of DCs after recognition of bacterial structural

components. These receptors, belonging to the Toll-like receptor family, are

differentially expressed on DC subpopulations and contribute to generate

functional diversity. To conclude this general part on DC function, there is

a description on how bacterial antigens are handled, processed and presented

by DCs.

In the second section, attention switches to the role of DCs in the

initiation and orchestration of innate immune responses. The section begins

describing how dendritic cells can directly participate in the uptake of

bacteria across mucosal surfaces and its consequences in terms of DC

activation. After microbial recognition, DCs act first as innate immune cells

that release inflammatory mediators that can strengthen and amplify the

innate immune response. In particular a novel monocyte-derived DC

population called TipDCs that produces large amounts of tumor necrosis

factor (TNF) and inducible nitric oxide synthase (iNOS) is reported. Then

DCs can leave the infected site to reach the draining lymph node for T cell

activation. Thus, DCs represent a link between innate and adaptive

immunity because their activation can lead on one side to the recruitment

and activation of innate immune cells like granulocytes, macrophages and

natural killer (NK) cells and on the other side to the activation of adaptive

immune cells. To achieve this, DCs can act on their own or in concert with

other innate immune cells like NK cells, as discussed in the last chapter of

this section.

The following section deals with the initiation of adaptive immune

responses that is conducted by DCs that have deciphered and integrated

signals deriving from the bacteria, the infected tissue and the recruited

immune cells. Two major examples of DC handling of strictly or facultative

intracellular bacteria have been considered, namely Legionella and

Salmonella. It is described how differently from macrophages, DCs have

evolved strategies to handle and control intracellular growth of Legionella and

to activate effective adaptive immune responses to control bacterial infection.

Interestingly, DCs can present bacterial antigens also when they are non-

infected after phagocytozing infected cells. This process also known as cross-

presentation is unique to DCs and favors the activation of T cell responses

toward Salmonella, Listeria and Mycobacterium.

Finally, strategies developed by bacteria to evade DC recognition and

activation are discussed in the fourth section. Here pathogen recognition

receptors are thoroughly discussed as possible targets for pathogens to

modulate immune function of antigen presenting cells. It is described that

the cross-talk between different classes of pathogen recognition receptors

can lead to suppression or activation of immune responses. In the following

chapter the ability of bacteria or their products to suppress the immune

response through the skewing of T cell responses toward regulatory T cells

or to subtypes which are inappropriate for bacterial elimination is reported.

�

x

PR

EF

AC

E

A major drawback of improper bacterial handling can result in chronic

inflammatory responses particularly at sites continuously exposed to bacteria

like the gut. Here, commensal bacteria are beneficial to the host as they help

digesting ingested food through the degradation of complex sugars and

metabolites. In order to tolerate ‘‘good’’ bacteria, the immune system has

developed strategies to cohabitate with beneficial bacteria and discriminate

harmful pathogens. When these strategies are disrupted, inflammatory

responses can arise leading to inflammatory bowel disease as discussed in

the last chapter of this section.

In conclusion, this book has brought together experts in several fields

of dendritic cell�bacteria interaction from their capture and recognition to

their killing, processing and induction of adaptive immunity. Much attention

has been focused on the tissue context where bacteria are handled by DCs.

When defects either in bacterial handling or in the interaction with the

environment are encountered, chronic infection or inflammation can arise.

�xi

PR

EF

AC

E

Abbreviations

APC antigen-presenting cell

ASK apoptosis signal-regulating kinase

BCG bacillus Calmette-Guerin

BIR baculoviral inhibitors of apoptosis repeat

CARD caspase recruitment domain

CD Crohn’s disease

cDC conventional DC

CLP common lymphoid progenitor

CLR C-type lectin-related

CMP common myeloid progenitor

CRD carbohydrate-recognition domain

CT cholera toxin

CTL cytotoxic T lymphocytes

DALIS dendritic cells aggresome-like induced structures

DC dendritic cell

DRIP defective ribosomal product

dsRNA double-stranded RNA

DSS dextran sodium sulfate

EC epithelial cell

ER endoplasmic reticulum

ERAD ER-associated degradation

ERAP endoplasmic reticulum aminopeptidase

FADD Fas (TNFRSF6)-associated via death domain

FAE follicle-associated epithelium

GALT gut associated lymphoid tissue

GFP green fluorescent protein

GM-CSF granulocyte-macrophage colony-stimulating factor

HCV Hepatitis C virus

HLA human leukocyte antigen

IAP inhibitors of apoptosis

IBD inflammatory bowel disease

IDC immature DC

IE-DAP g-d-glutyl-meso diaminopimelic acid

IFN interferon

Ii invariant chain

IKK IkB kinase

IL interleukin

iNOS inducible nitric oxide synthase

IRAK IL-1R-associated kinase

IRF interferon regulatory factor

ISGF IFN-stimulated gene factor

ISRE IFN-stimulated regulatory element

ITAM immunoreceptor tyrosine-based activation motif

JNK c-Jun N-terminal kinase

KIR killer Ig-like receptors

LAM lipoarabinomannan

LLO listeriolysin O

LP lamina propria

LPS lipopolysaccharide

LRR leucine-rich repeat

LTA lipoteichoic acid

mAB monoclonal antibody

MAL MyD88 adaptor-like

MAPKK mitogen activated protein kinase kinase

MAPKKK mitogen activated protein kinase kinase kinase

MDP muramyl dipeptide

MEF mouse embryonic fibroblast

MHC major histocompatibility complex

MLN mesenteric lymph nodes

NCR nitrogen catabolite repressor

NDV Newcastle disease virus

NEMO NF-kB essential modulator

NF nuclear factor

NK natural killer

NOD nucleotide-binding oligomerization domain

Nod-LRR nucleotide oligomerization domain-leucine-rich repeat

OVA chicken ovalbumin

�

xiv

AB

BR

EV

IAT

ION

S

PAMP pathogen associated molecular patterns

pDC plasmacytoid DC

PGN peptidoglycan

PI3P phosphoinositol-3-phosphate

PKR protein kinase R

PP Peyer’s patches

PPAR peroxisome-proliferator-activated receptor

PRR pathogen recognition receptor

RICK Rip-like interacting caspase-like apoptosis-regulatory protein

kinase

RIG retinoic acid-inducible protein

RIP receptor interacting protein

SARM sterile a and HEAT-Armadillo motif

siRNA small interfering RNA

SLE systemic lupus erythematosus

SPI Salmonella pathogenicity island

ssRNA single-stranded RNA

STAT signal transducer and activator of transcription

TAB tubulin antisense-binding protein

TAK TGFb-activating kinase

TAP transporter associated with antigen processing

TBK TANK-binding kinase

TGF transforming growth factor

TipDC tumor infiltrating pDC

TIR Toll/IL1 receptor

TIRAP TIR domain-containing adaptor protein

TJ tight junction

TLR Toll-like receptor

TNF tumor necrosis factor

TRAM TRIF-related adaptor molecule

TRIF TIR domain-containing adaptor inducing IFNbTSLP thymic stromal lymphopoietin

VSV Vesicular stomatis virus

�xv

AB

BR

EV

IAT

ION

S

Contributors

Shizuo Akira

Department of Host Defense

Research Institute for Microbial

Diseases

Osaka University

3-1 Yamada-oka

Suita

Osaka 565-0871

Japan

and

ERATO, Japan Science and

Technology Agency

3-1 Yamada-oka

Suita

Osaka 565-0871

Japan

Carlos Ardavın

Department of Immunology and

Oncology

Centro Nacional de Biotecnologia/

CSIC

Universidad Autonoma

28049 Madrid

Spain

Christoph Becker

I Department of Medicine

University of Mainz

55131 Mainz

Germany

Laurence Bougneres-Vermont

Institut Curie

Inserm u653

26 rue d’Ulm

75248 Paris

cedex 05

France

Miriam T. Brady

Immune Regulation Research

Group

School of Biochemistry and

Immunology

Trinity College

Dublin 2

Ireland

Anneke Engering

Department of Molecular

Cell Biology and

Immunology

VU Medical Center

v.d. Boechorststraat 7

1081 BT Amsterdam

The Netherlands

Guido Ferlazzo

Istituto Nazionale Ricerca sul

Cancro

Genoa

Italy

and

University of Messina

Messina 98100

Italy

Teunis B.H. Geijtenbeek

Department of Molecular Cell

Biology and Immunology

VU Medical Center

v.d. Boechorstraat 7

1081 BT Amsterdam

The Netherlands

Pierre Guermonprez

Institut Curie, inserm u653

26 rue d’Ulm

75248 Paris

cedex 05

France

Estella A. Koppel

Department of Molecular Cell

Biology and Immunology

VU Medical Center

v.d. Boechorststraat 7

1081 BT Amsterdam

The Netherlands

Peter McGuirk

Opsona Therapeutics

Biotechnology Building

Trinity College

Dublin

Ireland

Kingston H.G. Mills

Immune Regulation Research

Group

School of Biochemistry and

Immunology

Trinity College

Dublin 5

Ireland

Catarina Nogueria

Section of Microbial Pathogenesis

Yale University School of Medicine

Boyer Center for Molecular Medicine

295 Congress Avenue

New Haven

CT 06536

Eric G. Pamer

Infectious Diseases Service

Memorial Sloan-Kettering Cancer

Center

Immunology Program

Sloan Kettering Institute

1275 York Avenue

New York

New York 10021

USA

Maria Rescigno

European Institute of Oncology

Department of Experimental

Oncology

Via Ripamonti 435

20141 Milan

Italy

�

xviii

CO

NT

RIB

UT

OR

S

Craig R. Roy

Section of Microbial Pathogenesis

Yale University School of Medicine

Boyer Center for Molecular

Medicine

295 Congress Avenue

New Haven

CT 06536

USA

Natalya V. Serbina

Infectious Diseases Service

Memorial Sloan-Kettering Cancer

Center

Immunology Program

Sloan Kettering Institute

1275 York Avenue

New York

New York 10021

USA

Sunny Shin

Section of Microbial Pathogenesis

Yale University School of Medicine

Boyer Center for Molecular

Medicine

295 Congress Avenue

New Haven

CT 06536

USA

Osamu Takeuchi

Department of Host Defense

Research Institute for Microbial

Diseases

Osaka University

and

ERATO,

Japan Science and Technology

Agency

3-1 Yamada-oka

Suita

Osaka 565-0871

Japan

Yvette van Kooyk

Department of Molecular Cell

Biology and Immunology

VU Medical Center

v.d. Boechorststraat 7

1081 BT Amsterdam

The Netherlands

Sandra J. van Vliet

Department of Molecular Cell

Biology and Immunology

VU Medical Center

v.d. Boechorststraat 7

1081 BT Amsterdam

The Netherlands

Mary Jo Wick

Department of Microbiology and

Immunology

Goteborg University

Box 435

SE 40530 Goteborg

Sweden

�xix

CO

NT

RIB

UT

OR

S

PART I Dendritic cells and their rolein immunity

CHAPTER 1

Subpopulations and differentiation of mousedendritic cells

Carlos ArdavınUniversidad Autonoma

1.1 DENDRITIC CELL SUBPOPULATIONS

Dendritic cells (DCs) have an essential function in the immune system by

participating in primitive defense responses that constitute the innate

immunity, as well as in the induction and regulation of antigen-specific

immune responses. This allows DCs to control infections caused by parasitic

and microbial pathogens, to block tumour growth and to exert a precise

regulation of T cell, B cell and NK cell immune responses. In addition, DCs

also fulfill a pivotal role in the induction and maintenance of Tcell tolerance.

The functional diversity characterizing the DC system relies essentially on

the remarkable plasticity of the DC differentiation process, which dictates the

acquisition of DC functional specialization through the generation of a large

collection of DC subpopulations (reviewed by Shortman and Liu, 2002).

Dendritic cells are located both in the lymphoid organs (such as the spleen

or the lymph nodes), and in non-lymphoid tissues (such as the skin or the

liver), and can be classified in two major categories: conventional DCs

(cDCs), and plasmacytoid DCs (pDCs). Whereas in turn cDCs comprise

multiple DC subpopulations endowed with specific functions, little is known

about the functional heterogeneity of pDCs. A summary of the most relevant

phenotypic and functional characteristics of the main DC subpopulations

present in mice is shown in Table 1.1.

A first group of cDCs includes those that are common, and largely

restricted, to the majority of organized lymphoid organs of the immune

system, and perform their specific functions, as immature or mature

DCs, within these organs. This group of lymphoid organ-restricted cDCs

comprises three main DC subpopulations, namely CD8þ CD11b� DCs

(herein called CD8þ DCs), CD8� CD11bþ DCs (herein called CD8�

DCs), and CD8� CD11b� DCs (herein called CD11b� DCs). CD8� DCs

Tab

le1

.1.Location

andphenotypiccharacteristicsoftheprincipalm

ouseDCsubpopulations

pDCs

CD8þ

DCs

CD8�

CD4þ

DCs

CD8�

CD4�

DCs

CD11b�

DCs

Langerhan

scells

Dermal

DCs

Location

Th

ymu

sY

esY

esN

oY

esa

Few

No

No

Sp

leen

Yes

Yes

Yes

Yes

No

No

No

Sk

inb

No

No

No

No

Yes

Yes

Per

iph

eral

LN

sY

esY

esF

ewY

esF

ewY

esY

es

Inte

stin

alP

eyer

’s

pat

ches

Yes

Yes

Few

Yes

Yes

No

No

Mes

ente

ric

LN

sY

esY

esF

ewY

esY

esN

oc

Oth

erlo

cati

on

sB

on

e

mar

row

,

blo

od

,

lun

g

Liv

erL

iver

Liv

erE

pid

erm

is,

inte

stin

al

S-E

,lu

ng

S-E

,

vag

ina

S-E

Inte

stin

alL

-P,d

bro

nch

ial

L-P

,d

lun

gin

ters

titi

um

,d

live

rp

aren

chym

a,d

kid

ney

par

ench

yma,d

CN

Sp

aren

chym

ad

Phen

otype

CD11c

þin

tþ

hig

hþ

hig

hþ

hig

hþ

hig

hþ

hig

hþ

hig

h

CD11b

�þ

low

þh

igh

þh

igh

�þ

hig

hþ

hig

h

CD8

þe

þ�

��

�(!

þ)f

�

CD4

þe

�þ

��

��

B220

þ�

��

��

�

CD62L

þ�

��

Not an

alyz

ed

��

DE

C-205

�þ

hig

h�

��

þh

igh

þlo

w

Ab

bre

viat

ion

s:C

NS

,ce

ntr

aln

ervo

us

syst

em;

LN

s,ly

mp

hn

od

es;

L-P

,la

min

ap

rop

ria;

S-E

,st

rati

fied

epit

hel

ium

.

Notes

aT

hym

icC

D8�

DC

sh

ave

bee

nre

po

rted

toex

pre

sslo

wle

vels

of

CD4,

alth

ou

gh

ith

asb

een

sug

ges

ted

that

infa

ctC

D8�

thym

icD

Cs

are

CD4�

,si

nce

CD4

app

ears

tob

ep

ick

edu

pb

yth

ymic

DC

sfr

om

surr

ou

nd

ing

CD4þ

Tce

lls.

bp

DC

sar

eab

sen

tfr

om

the

skin

inst

ead

yst

ate,

bu

tca

nb

ere

cru

ited

toth

islo

cati

on

du

rin

gin

flam

mat

ion

.c

DC

sw

ith

sim

ilar

char

acte

rist

ics

than

tho

sed

efin

ing

der

mal

DC

saf

ter

mig

rati

on

toth

ep

erip

her

alL

Ns

are

fou

nd

inth

em

esen

teri

cL

Ns;

thes

ece

lls

hav

eb

een

clai

med

toco

rres

po

nd

toin

test

inal

L-P

DC

s.d

Inte

rsti

tial

DC

sth

ath

ave

bee

nsu

gg

este

dto

be

fun

ctio

nal

lyre

late

dto

der

mal

DC

sh

ave

bee

nd

escr

ibed

inth

ese

loca

tio

ns.

ep

DC

sh

ave

bee

nd

emo

nst

rate

dto

up

reg

ula

teb

oth

CD8

and

CD4

du

rin

gac

tiva

tio

n.

fL

ang

erh

ans

cell

sh

ave

bee

nd

emo

nst

rate

dto

up

reg

ula

teC

D8

du

rin

gin

vivo

mat

ura

tio

nan

dm

igra

tio

nfr

om

the

skin

toth

ed

rain

ing

lym

ph

no

des

.

can be in turn subdivided in CD8� CD4þ DCs and CD8� CD4� DCs.

Whereas CD8þ DCs and/or CD8� DCs are present in the thymus, spleen,

lymph nodes and lymphoid tissue of the intestinal and respiratory tracts

(reviewed by Shortman and Liu, 2002), the CD11b� DC subpopulation

appears to be predominantly related to the intestinal lymphoid system

(reviewed by Johansson and Kelsall, 2005), representing approximately one-

third of the DCs found in the Peyer’s patches and mesenteric lymph nodes.

CD8þ DCs and CD8� DCs do not appear to migrate or recirculate to other

effector organs of the immune system to fulfill their functions. In this

sense, CD11b� DCs have been tentatively included in this category since

they appear to be present in the majority of lymphoid organs (reviewed by

Johansson and Kelsall, 2005), but their immunobiology, and particularly

their migratory behavior, remains largely unknown.

A second group of cDCs comprises those located, in an immature state,

in antigen-uptake sites within non-lymphoid organs. Upon contact with an

antigen, these DCs migrate through the lymph vessels to the lymph nodes,

where they interact with antigen-specific Tcells, and in some cases with other

effector cells of the immune system, including DCs, NK cells and B cells.

This group of migrating-cDCs comprises Langerhans cells (located in

the epidermis, and other stratified and pseudo-stratified epithelia of the

intestinal, respiratory and reproductive tracts), and interstitial DCs. These in

turn include dermal DCs, and other interstitial DC subpopulations, as those

found in the lamina propria of the intestinal and respiratory tracts, as well as

those located in the lung interstitium, and in the parenchyma of the liver,

kidney and CNS.

A number of experimental evidences suggest that in the steady state

both lymphoid-organ restricted cDCs and migrating-cDCs are generated

locally from blood-borne, immediate DC precursors, originating in the

bone marrow (reviewed by Ardavın, 2003). Interestingly, during ongoing

immune responses, a strong increase in the absolute number of both

lymphoid organ-restricted cDCs and migrating-cDCs has been reported,

raising the problem of the identity of the immediate DC precursors

responsible for the generation of these de novo-formed DCs. It could be

hypothesized that the same precursors are responsible for the generation of

cDCs under steady state and infection. Alternatively, during infection, apart

from, or instead of the precursors functioning in steady state, additional

inflammatory precursors, with equivalent or complementary DC differentia-

tion potential, could be recruited. Although this remains an open issue in DC

biology, different research groups have reported that during inflammation

and/or infection, monocytes, which represent so far the best known DC

�

6

C.

AR

DA

VIN

precursors, could be recruited to both peripheral tissues (such as the dermis)

and to lymphoid organs, and differentiate locally into DCs (reviewed by

Leon et al., 2005). The relationships between monocyte-derived DCs (or

other putative de novo-formed inflammatory DCs) and the pre-existing

lymphoid organ-restricted cDCs or migrating-cDC subpopulations, has to be

addressed. In this sense, data from our laboratory suggest that during

infection with Leishmania major, monocytes are actively recruited to the

popliteal lymph nodes draining the site of infection, where they differentiate

into CD11bþ CD8� DCs, with phenotypic characteristics that differ from

those defining the DC subpopulations pre-existing in the popliteal lymph

nodes (C. Ardavın et al., unpublished data). Although the role played by

monocyte-derived DCs during Leishmania infection has to be defined, our

results support the concept that the immune responses against certain

pathogens could involve the participation of newly-formed DCs endowed

with specific functions.

In contrast to cDCs, pDCs appear to differentiate in the bone marrow,

and migrate to the blood and lymphoid organs, where they are activated

and participate in anti-viral immune responses (reviewed by Colonna

et al., 2004). In addition, it has been shown that pDCs can be recruited to

the inflamed skin during atopic dermatitis, contact dermatitis or psoriasis,

and to systemic lupus erythematosis (SLE)-associated skin lesions

(reviewed by Valladeau and Saeland, 2005). However, the possibility that

during inflammation and/or infection pDC precursors are recruited to the

lymphoid organs and differentiate locally, has not been addressed in depth

and therefore cannot be excluded.

An integrated model of the development and function of the mouse DC

system is represented in Figure 1.1.

1.1.1 Lymphoid organ-restricted cDCs

In the steady state, CD8þ DCs, CD8� DCs and CD11b� DCs are found in the

lymphoid organs as immature DCs; however, under situations of inflamma-

tion and/or infection DCs enter into a maturation program as a result of the

engagement of DC activating receptors (reviewed in Johansson and Kelsall,

2005 and Villadangos and Heath, 2005). Globally, these DC subpopulations

are considered to induce T cell responses after capturing antigens that gain

access to the lymphoid organs through the blood or lymph vessels. In

addition, as discussed below, at least CD8þ DCs can also cross-present

antigens captured in the vicinity of epithelial surfaces by migrating DCs that

�7

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

Fig

ure

1.1

.In

teg

rate

dm

od

elo

fth

ed

evel

op

men

tan

dfu

nct

ion

of

the

mo

use

DC

syst

em.

Bla

ckso

lid

arro

ws

rep

rese

nt

dif

fere

nti

atio

nan

d/o

rm

atu

rati

on

pro

cess

es;

bla

ckb

rok

enar

row

sre

pre

sen

tm

igra

tio

np

roce

sses

;g

rey

bro

ken

arro

ws

rep

rese

nt

hyp

oth

etic

ald

iffe

ren

tiat

ion

or

mig

rati

on

pro

cess

es.B

lack

thic

k

arro

ws

ind

icat

eth

ein

du

ctio

no

fD

Cac

tiva

tio

nin

itia

ted

by

pat

ho

gen

so

rin

flam

mat

ory

med

iato

rs.W

hit

eth

ick

arro

ws

ind

icat

ean

tig

entr

ansf

er.D

ou

ble

arro

ws

ind

icat

eT

cell

dir

ectp

rim

ing

;gre

yth

ick

arro

ws

ind

icat

eT

cell

cro

ssp

rim

ing

.Ab

bre

viat

ion

s:i-

DC

,im

mat

ure

DC

;LC

,Lan

ger

han

sce

ll;m

-DC

,mat

ure

DC

.(F

or

a

colo

ur

vers

ion

of

this

fig

ure

,p

leas

ere

fer

toth

eco

lou

rin

sert

bet

wee

np

ages

12

and13

.)

transport these antigens to the lymph nodes and transfer them to CD8þ DCs

(Belz et al., 2004).

Maturation of lymphoid organ-restricted DCs could involve a local

migration process, from the antigen capture zones to the T cell areas within

the same lymphoid organ, but not the migration between different organs

through the lymphatics. In particular, it has been reported that upon

activation, CD8� DCs migrate from the outer to the inner part of the splenic

white pulp (De Smedt et al., 1996; Reis e Sousa et al., 1997), and from the

subepithelial dome to the interfollicular region of the Peyer’s patches

(Iwasaki and Kelsall, 2000). In general, antigen presentation by immature

CD8þ DCs or CD8� DCs leads to T cell tolerance, whereas antigen uptake by

these DC subpopulations in the presence of maturation stimuli leads to the

induction of T cell immunity by mature CD8þ DCs or CD8� DCs (reviewed

by Steinman and Nussenzweig, 2002).

CD8þ DCs

In steady state, CD8þ DCs are located in the cortico-medullary zone and

medulla of the thymus (reviewed by Ardavın, 1997) (where they participate

in the negative selection of the developing thymocytes) and in the T cell

areas of the lymphoid organs (reviewed by Villadangos and Heath, 2005)

(i.e. inner part of the splenic white pulp, deep cortex of the lymph nodes,

and interfollicular regions of the lymphoid aggregates of the intestinal

tract). CD8þ DCs are less efficient than CD8� DCs in capturing antigens

by endocytosis or phagocytosis (Leenen et al., 1998). However, they display a

high capacity to internalize apoptotic cells (Iyoda et al., 2002), which has been

correlated with their ability to induce peripheral tolerance to self tissue-

associated antigens in the steady state (Belz et al., 2002; Liu et al., 2002).

CD8þ DCs can stimulate CD4þ or CD8þ T cell responses by direct- or

cross-priming, after antigen uptake in the lymphoid organs (reviewed by

Villadangos and Heath, 2005). Alternatively, it has been proposed that after

antigen uptake in non-lymphoid organs by dermal or interstitial DCs,

which subsequently migrate to the lymph nodes, these antigens can be cross-

presented by lymph node CD8þ DCs, as the result of antigen transfer from

the migrating-DCs to resident CD8þ DCs (reviewed by Villadangos and

Heath, 2005). In this regard, results from various research groups agree on

the idea that CD8þ DCs are the most effective, if not the sole, DC

subpopulation capable of stimulating a T cell response by cross-presentation

in vivo (den Haan et al., 2000; Pooley et al., 2001), although it has been

recently reported that Langerhans cells can stimulate OVA-specific T cells

in vivo by cross-priming (Mayerova et al., 2004). The cross-priming capacity

�9

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

of CD8þ DCs has been proposed to rely on their potential to process exo-

genous antigens to direct them to the MHC I pathway (den Haan et al., 2000;

Pooley et al., 2001) and/or on the ability of this DC subset to internalize cell-

associated antigens (Schulz and Reis e Sousa, 2002).

Data derived from different experimental systems support the concept

that CD8þ DCs are efficient producers of interleukin (IL)-12, and are mainly

involved in the induction of Th1 responses (reviewed in Iwasaki and

Medzhitov, 2004). In this sense, it has been proposed that CD8þ DCs play

an essential role in the induction of CD8þ T cell responses, particularly

during infection by both cytolytic and noncytolytic viruses (Belz et al., 2004,

2005), although recent data indicate that they are also involved in the

initiation of T cell immunity against intracellular bacteria (Belz et al., 2005).

On the other hand, CD8þ DC involvement in the induction of anti-protozoan

parasite immunity is controversial. Whereas CD8þ DCs have been demon-

strated to play an essential role in the induction of Th1 responses against

Toxoplasma gondii (Aliberti et al., 2000), they do not appear to participate in

anti-Leishmania major Th1 protective responses (Filippi et al., 2003; Ritter

et al., 2004).

CD8� DCs

In contrast to CD8þ DCs, CD8� DCs appear to be mainly located adjacent to

the principal antigen entry zones of the lymphoid organs, i.e. the outer part

of the splenic white pulp in association with the marginal zone,

the subsinusal layer beneath the lymph node subcapsular sinus and the

subepithelial area of the lymphoid tissue of the intestinal tract (reviewed

by Villadangos and Heath, 2005). In the spleen, around two-thirds of

CD8� DCs are CD4þ, whereas in the Peyer’s patches and both in the

peripheral and mesenteric lymph nodes the vast majority of CD8� DCs

correspond to the CD4� subset (reviewed by Johansson and Kelsall, 2005).

However, it has been reported that during infection by the mouse

mammary tumor virus, there was a progressive increase in the proportion

of CD4þ DCs among the CD8� DC subpopulation located in the popliteal

lymph nodes draining the infection area (Martin et al., 2002).

CD8� DCs display a high endocytic and phagocytic capacity, but are

inefficient in capturing apoptotic cells. In this sense, it has been reported that

CD8� DCs are responsible for the in vivo stimulation of CD4þ T cells after

immunization with antigen-loaded DC-derived exosomes (Thery et al., 2002).

Data on the role of CD8� DCs in the induction of T cell responses in vivo are

limited, and in particular those addressing differentially the function of

CD8� CD4þ DCs versus CD8� CD4� DCs. Globally, results derived from

�

10

C.

AR

DA

VIN

in vivo, but principally in vitro experiments performed with splenic CD8�

DCs support the concept that, in contrast to their CD8þ counterparts, CD8�

DCs appear to be poor IL-12 producers, and would be mainly involved in the

induction of Th2 responses (reviewed in Iwasaki and Medzhitov, 2004). In

addition, CD8� DCs from the Peyer’s patches (belonging to the CD4� subset)

have been demonstrated to produce high amounts of IL-10, and to induce

Th2 responses leading to IgA production (Iwasaki and Kelsall, 1999; Sato

et al., 2003). Moreover, these cells can also induce the differentiation of

IL-10 producing T regulatory cells (Iwasaki and Kelsall, 2001). However

the Th1 versus Th2-polarizing potential of CD8� DCs remains controversial,

and therefore needs to be addressed carefully, because it has been reported

that CD8� DCs can produce IL-12 in the presence of anti-IL-10 (Fallarino

et al., 2002; Maldonado-Lopez et al., 2001) in response to a-galactosylcer-

amide (Fujii et al., 2003) and Toll-like receptors (TLR)-7 ligands (Edwards

et al., 2003).

On the other hand, a role for CD8� DCs in the induction of protective

immunity against Leishmania major has been proposed by Filippi et al.

(2003), who reported that CD11bþ CD8� DCs located in the lymph nodes

draining the site of infection were responsible for the induction of Lack-

specific Th1 responses. However, a more detailed phenotypic and functional

analysis of these DCs is required to demonstrate conclusively whether these

CD11bþ CD8� DCs actually correspond to those CD8� DCs pre-existing in

the lymph node prior to the onset of Leishmania infection. Alternatively, these

CD11bþ CD8� DCs could represent dermal DCs that had migrated from the

skin as proposed by Iwasaki (2003), or to newly-formed monocyte-derived

DCs that can be also described as CD11bþ CD8� DCs. In this sense, dermal

DCs and monocyte-derived DCs, but not CD8� DCs, express DEC-205 (Henri

et al., 2001) and Ly-6C (C. Ardavın et al., unpublished data), respectively.

These data suggest that under certain physiological conditions

CD8� DCs could participate in the induction of Th1 responses. On the

other hand, discrepancy between published data could rely on the yet

undefined differential functions of the CD4þ versus CD4� subsets of

CD8� DCs, because the majority of the experiments addressing the role

of CD8� DCs have been carried out with unfractionated CD8� DCs.

CD11b� DCs

CD11b� DCs are preferentially found in the Peyer’s patches and mesenteric

lymph nodes, and represent only a minor subset in the spleen and peripheral

lymph nodes; in addition they are the principal DC subpopulation in the liver

(reviewed by Johansson and Kelsall, 2005). Within the Peyer’s patches,

�11

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

CD11b� DCs are located both in the subepithelial dome and the inter-

follicular regions; in addition immature CD11b� DCs appear to be present

within the follicular-associated epithelium. In the mesenteric lymph nodes

CD11b� DCs are located in the T cell areas.

In contrast to IL-10 producing intestinal CD8� DCs, CD11b� produced

IL-12 after microbial stimulation and induce Th1-polarized responses

(Iwasaki and Kelsall, 2001). In addition, recent data from Dr. Kelsall’s

laboratory demonstrate that CD11b� DCs can internalize and process

antigens from type 1 Reovirus-infected Peyer’s patch epithelial cells, and

stimulate virus-specific T cells by cross-priming (Fleeton et al., 2004).

1.1.2 Migrating cDCs

Langerhans cells, dermal DCs and other interstitial DC subpopulations are

strategically located in close association with epithelial surfaces that are

exposed to microbial pathogens. They constitute a group of DC subpopula-

tions expressing specialized endocytic and phagocytic receptors allowing

a highly efficient uptake and processing of pathogen-derived antigens

(reviewed by Valladeau and Saeland, 2005). In addition, it has been

demonstrated that lamina propria-specific DCs can extend dendrites

between epithelial cells and sample microorganisms from the intestinal

lumen (Rescigno et al., 2001).

Recent data suggest that migrating-cDC subpopulations display a

constitutive rate of migration to the lymph nodes after antigen uptake in

the absence of inflammatory signals that is generally associated with the

induction of T cell tolerance. However, whereas this concept appears to be

true for dermal or interstitial DCs (Itano et al., 2003; Scheinecker et al., 2002;

Turley et al., 2003), a recent study supports that Langerhans cells can activate

naive T cells upon migration to the lymph nodes, in the steady state

(Mayerova et al., 2004). Nevertheless, as described for CD8þ DCs and

CD8� DCs, if antigen capture occurs in the presence of inflammatory stimuli

such as TNF-a or IL-1b, or TLR-ligands, migrating-cDCs undergo a CCR7-

dependent process of migration accompanied by maturation that leads to

the induction of T cell immunogenic responses in the draining lymph nodes

(reviewed by Valladeau and Saeland, 2005). However, although a series of

reports support the view that both Langerhans cells and dermal or interstitial

DCs can fulfill this function, recent data challenge this paradigm of DC

immunobiology. In this sense, experimental evidence based on the induction

of T cell responses in vivo suggest that Langerhans cells may not have

an important role in the capture, transport and presentation of skin

�

12

C.

AR

DA

VIN

Fig

ure

1.1

.In

teg

rate

dm

od

elo

fth

ed

evel

op

men

tan

dfu

nct

ion

of

the

mo

use

DC

syst

em.

Fig

ure

1.2

.M

od

elo

fth

ere

gu

lati

on

of

mo

use

DC

dif

fere

nti

atio

n.

Fig

ure

4.1

.M

ech

anis

ms

of

bac

teri

alu

pta

ke.

Fig

ure

4.2

.T

he

cro

ss-t

alk

bet

wee

nE

Cs

and

DC

sin

Salmonella

typhim

urium

han

dli

ng

.

pathogen-derived antigens to T cells, despite their capacity to internalize a

large variety of microbial pathogens (reviewed by Villadangos and Heath,

2005). This function would be rather carried out essentially by dermal DCs or

interstitial cDCs, as demonstrated during infection by Leishmania major

(Ritter et al., 2004) or Herpes simplex virus type 2 (Iwasaki, 2003). On the other

hand, it has been reported that in certain viral infection models, neither

dermal nor interstitial cDCs that capture and transport antigens to the lymph

nodes, have an essential role in the induction of CD8þ T cell responses.

In this sense, it has been demonstrated that the CD8þ T cell response

against Herpes simplex virus type 1 was mediated by CD8þ DCs, to which the

antigen was transferred from interstitial-cDCs upon migration to the lymph

node (Allan et al., 2003). In a different experimental system, it has been

shown that during infection by Influenza or Herpes simplex virus type 1, virus-

derived antigens were presented to specific CD8þ T cells in the draining

lymph nodes, by both interstitial migrating-cDCs that have internalized

the virus in the lung, and by resident lymph node CD8þ DCs, to which

migrating-cDCs had transferred the antigen (Belz et al., 2004).

1.1.3 Plasmacytoid DCs

Murine pDCs can be characterized as CD11cþ CD11b� B220þ Ly-6Cþ cells,

and are found in the bone marrow, thymus, blood and all peripheral

lymphoid organs. In contrast to cDCs, pDCs appear to differentiate in the

bone marrow (at least under non-inflammatory and/or infectious condi-

tions), and express CD62L (L-selectin) that allows them to be recruited

through high endothelial venules to the lymph nodes and the lymphoid

tissue associated to the intestinal and respiratory tracts (reviewed by Barchet

et al., 2005). Interestingly, three pDC-specific monoclonal antibodies (mAbs)

have been developed recently and represent powerful tools to analyze the

development and function of pDCs. The mAb 120G8 (Asselin-Paturel et al.,

2003) recognizes a molecule that is expressed specifically by pDCs in non-

stimulated mice, but is upregulated by type I interferon (IFN) on other cell

types. The mAb 440c mAb (Blasius et al., 2004) specifically recognizes pDCs

in both naive and type I IFN-stimulated mice; interestingly pDCs produce

type I IFN after 440c-mediated engagement of its yet undefined counter-

receptor. Finally the mAb mPDCA-1 (Krug et al., 2004) specifically recognizes

pDCs. Treatment by either mPDCA-1 or 120G8 promotes pDC depletion in

vivo. On the other hand, based on the expression of Ly49Q, two pDC subsets

have been recently defined (Kamogawa-Schifter et al., 2005). Peripheral

pDCs are Ly49Qþ, whereas both Ly49Qþ and Ly49Q� pDCs exist in bone

�13

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

marrow, the latter being less efficient in producing inflammatory cytokines,

such as IL-6, IL-12 and TNF-a.

pDCs are characterized by their capacity to produce large amounts of

type I-IFN during viral infections that relies essentially in a TLR-dependent

mechanism initiated after virus endocytosis, and subsequent recognition of

viral nucleic acids by TLR-7 or TLR-9 in an acidic endosomal compartment

(Iwasaki and Medzhitov, 2004). The high type I IFN production efficiency

of pDCs appears to be in part related to their constitutive expression of

interferon regulatory factor (IRF)-7, allowing its rapid activation and

subsequent transcription of IFN-a genes, independently of previous IRF-3

activation (as described for IRF-7 induction in non-pDCs) (Barchet et al.,

2002; Prakash et al., 2005). Differential transport of viral nucleic acids into

endosomal versus lysosomal compartments in pDCs and cDCs, respectively,

could also contribute to the functional specialization of pDCs as type I IFN

producers.

Apart from providing this type I IFN-mediated initial defense mechan-

ism against viral infections after exposure to DC maturation stimuli, such

as TLR-7/9 ligands or CD40L, pDCs could also participate in the induction

of antigen-specific T cell responses (reviewed by Colonna et al., 2004),

although whether pDCs can activate naıve T cells in vivo after viral infection

remains controversial. However, pDCs have been demonstrated to induce

the proliferation of virus-specific, antigen-primed CD8þ T cells and Th1

CD4þ T cells (Krug et al., 2003; Schlecht et al., 2004). Based on these data,

it has been hypothesized that pDCs could synergize with cDCs in the

induction of Th1 responses during in vivo viral infections.

1.2 DENDRITIC CELL DIFFERENTIATION

Current knowledge on the origin and differentiation pathways leading to the

generation of DCs relies essentially on in vivo DC reconstitution experiments

in which defined hematopoietic precursors were transferred into irradiated

mice, and on in vivo and in vitro studies performed with mice deficient

in growing or transcription factors involved in DC differentiation (reviewed

by Ardavın, 2003). Due in part to the technical complexity and limitations

of such assays, the majority of these studies have focused almost exclusively

on the differentiation of CD8þ DCs, CD8� DCs and pDCs, and occasionally

on skin Langerhans cells. As a consequence, how the differentiation of other

DC subpopulations proceeds remains essentially unknown. Nevertheless,

as discussed below, these experiments have provided valuable information

on the differentiation pathways generating DCs from early myeloid or

�

14

C.

AR

DA

VIN

lymphoid precursors. However, unfortunately they have so far not allowed

a comprehensive characterization of the immediate precursors of DCs that

home to specific effector sites where DCs exert their function. Figure 1.1

summarizes current knowledge on the development of DC subpopulations.

1.2.1 Myeloid and lymphoid DC differentiation pathways

DCs were originally thought to be derived exclusively from myeloid

precursors, but experiments showing that both thymic and splenic DCs

(particularly the CD8þ and CD8� DC subpopulations) can be originated

from early thymic precursors, devoid of myeloid reconstitution potential

(Ardavın et al., 1993), led to the hypothesis that DCs could be also generated

through a lymphoid differentiation pathway. This theory was reinforced by

additional data from experiments demonstrating that bone marrow common

lymphoid progenitors (CLPs) had the capacity to differentiate into the

same DC subpopulations as those generated experimentally by early thymic

precursors (Traver et al., 2000). Subsequent studies, performed by co-

transferring CLPs and common myeloid progenitors (CMPs) into irradiated

recipients, indicated that, at least under these experimental conditions, the

differentiation of CD8þ DCs and CD8� DCs in the thymus and spleen was

the result of a simultaneous contribution of CLPs and CMPs (Manz et al.,

2001; Wu et al., 2001). The concept that certain DC subpopulations can

be generated through both the myeloid and the lymphoid differentiation

pathways was recently extended to the pDC subpopulation, by demonstrating

that both CMPs and CLPs can give rise to pDCs after intravenous transfer

(D’Amico and Wu, 2003) or in Flt3L-driven cultures (Karsunky et al., 2005).

Although so far the developmental derivation of organ-specific DC

subpopulations has not been addressed in depth, globally these data support

the hypothesis that both myeloid and lymphoid precursors are endowed with

the capacity to differentiate into diverse DC subpopulations. However,

whether these experiments reflect the physiological situation, i.e. whether

in fact both differentiation pathways actually contribute to the generation

of the different DC subpopulations, and if so, what is the differential

contribution of CMPs and CLPs to the generation of each DC subpopulation,

remains essentially unknown. In this regard, the model of the dual contri-

bution of the myeloid and lymphoid pathways to DC development appears

to be particularly controversial with regard to the differentiation of pDCs.

This DC subpopulation has been claimed to derive from lymphoid precur-

sors under physiological conditions, based on a series of studies performed

both in humans and mice, describing that pDCs express several molecules

�15

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

closely related to the lymphoid lineage, such as pTa, spi-B, IL-7R, PIII CIITA,

and have IgH gene rearrangements (reviewed by Colonna et al., 2004).

However, these data do not allow one to draw a definitive conclusion on pDC

origin because two recently published reports support the view that in fact

human and mouse pDCs can activate a lymphoid genetic program

irrespective of whether they are derived from lymphoid or myeloid

progenitors (Chicha et al., 2004; Shigematsu et al., 2004).

1.2.2 Control of DC differentiation

Interestingly, the fact that the DC differentiation capacity of CMPs and CLPs

is largely associated with the Flt3-positive fraction of these progenitor

populations (D’Amico and Wu, 2003) suggests that the DC differentiation

potential of myeloid and lymphoid hematopoietic precursors is determined

by the expression of the receptor for Flt3L. The cytokine Flt3L plays a crucial

role in DC development, as demonstrated in Flt3L-deficient mice displaying

a profound defect in the differentiation of all the DC subpopulations that

were analyzed (McKenna et al., 2000). Although both in vivo and in vitro

evidence indicate that Flt3L determines the commitment of CMPs and

CLPs toward DC differentiation, and drives the early steps of this process

(reviewed by Ardavın, 2003), a number of experimental evidences suggest

that additional cytokines are selectively required to induce and direct the

generation of specific DC subpopulations. Unfortunately, data currently

available on this issue are far from allowing the establishment of a

comprehensive model of cytokine-mediated regulation of the differentiation

of mouse DC subpopulations. In this regard, although as discussed below,

a number of cytokines appear to be involved in defined pathways of DC

differentiation, the cytokine combinations controlling the development of

each DC subpopulation remain to be defined.

In this regard, Flt3L is not only a key cytokine in driving the initial steps

of DC differentiation from CMPs and CLPs, but also appears to directly

control the generation of pDCs (Karsunky et al., 2005; Laouar et al., 2003).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been

demonstrated to drive the differentiation of mouse monocytes into DCs

(Leon et al., 2005), but its precise role on the generation of the different DC

subpopulations has not been addressed in depth. However, on the basis

of data derived from experiments of in vivo treatment with GM-CSF (Daro

et al., 2000), and from the analysis of IRF-4 and/or IRF-8-deficient

mice (Tamura et al., 2005), it has been proposed that GM-CSF and Flt3-L

control, through IRF-4 and IRF-8, the differentiation of CD8� DCs and CD8þ

�

16

C.

AR

DA

VIN

DCs, respectively. Moreover, GM-CSF appears to inhibit the differentiation of

pDCs (Gilliet et al., 2002). Mice deficient in LTa, or in its receptor LTb-R,

display a deficient splenic CD8� DC development, which has been correlated

with the ability of LTa to induce the proliferation of CD8� DCs (Kabashima

et al., 2005). Finally, both in vivo (Borkowski et al., 1996) and in vitro

(Zhang et al., 1999) assays have demonstrated the requirement of TGF-b for

the differentiation of epidermal Langerhans cells.

Studies published over the last few years have provided new insights

on the signaling pathways and transcriptional regulation associated with

the development of certain DC subpopulations (reviewed by Ardavın, 2003).

Several members of the IRF-family of transcription factors have been

proposed to control the differentiation of diverse DC subpopulations. In

this regard, the analysis of mice deficient in IRF-8 has demonstrated the

involvement of this transcription factor in the differentiation of CD8þ DCs

(Aliberti et al., 2003; Schiavoni et al., 2002), Langerhans cells (Schiavoni

et al., 2004) and pDCs (Schiavoni et al., 2002; Tsujimura et al., 2003), whereas

IRF-4 is required for the development of CD8� CD4þ splenic DCs (Suzuki

et al., 2004), and to a lesser extent of pDCs (Tamura et al., 2005). Finally, IRF-2

has been shown to participate in the differentiation of Langerhans cells

(Ichikawa et al., 2004), and splenic CD8� CD4þ DCs (Honda et al., 2004;

Ichikawa et al., 2004), whose dependence on IRF-2 has been claimed to rely

on the inhibition of IFN-a production that would in turn negatively control

the generation of CD8� CD4þ DCs.

In addition, a defective development of CD8� DCs has also been

reported in mice deficient in the adapter molecule TRAF-6 (involved in the

signaling pathway of IL-1, TNF-a and TLRs) (Kobayashi et al., 2003), and in

the transcription factors Ikaros C (Wu et al., 1997) and RelB (Wu et al., 1998).

Finally, the differentiation of splenic CD8þ DCs appears to require the Rho

family guanosine triphosphatases Rac 1 and Rac 2 (involved in DC

migration) (Benvenuti et al., 2004), and the transcription factor Id2

(Hacker et al., 2003), the latter being also involved in the differentiation of

epidermal Langerhans cells. Based on these data, a model of the control of

mouse DC differentiation is proposed in Figure 1.2.

Despite the relevance of these results, the specific cytokines that initiate

these signaling pathways and activate these transcription factors on

immediate DC precursors have to be precisely defined. Based on the current

literature on this subject, it can be hypothesized that the generation of

the different DC subpopulations is controlled not only by cytokines that

trigger specific differentiation programs on immediate DC precursors,

but also by cytokines and chemokines responsible for the correct

�17

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

Fig

ure

1.2

.M

od

elo

fth

ere

gu

lati

on

of

mo

use

DC

dif

fere

nti

atio

n.

Th

isfi

gu

resu

mm

ariz

esth

ecu

rren

tk

no

wle

dg

eo

nth

ep

rin

cip

alcy

tok

ines

(in

blu

e),

and

tran

scri

pti

on

fact

ors

and

adap

ter

mo

lecu

les

(in

red

)cl

aim

edto

con

tro

lth

ed

iffe

ren

tD

Cd

iffe

ren

tiat

ion

pat

hw

ays.

(Fo

ra

colo

ur

vers

ion

of

this

fig

ure

,p

leas

e

refe

rto

the

colo

ur

inse

rtb

etw

een

pag

es12

and13

.)

development of defined DC subpopulations, by allowing immediate DC

precursors to locate in specific environments. In this regard, future

research on DC development and acquisition of DC functional specialization

needs to be focused on the characterization of DC precursors because,

although recent research on DC development has provided important

insights on the origin and control of the differentiation pathways leading

to the different DC subpopulations, the identity of the immediate DC

precursors and how their differentiation is controlled has only begun to

be unravelled.

REFERENCES

Aliberti, J., Reis e Sousa, C., Schito, M., Hieny, S., Wells, T., Huffnagle, G. B. and

Sher, A. (2000). CCR5 provides a signal for microbial induced production

of IL-12 by CD8 alphaþ dendritic cells. Nat. Immunol., 1, 83–7.

Aliberti, J., Schulz, O., Pennington, D. J., Tsujimura, H., Reis e Sousa, C.,

Ozato, K. and Sher, A. (2003). Essential role for ICSBP in the in vivo

development of murine CD8alphaþ dendritic cells. Blood, 101, 305–10.

Allan, R. S., Smith, C. M., Belz, G. T., van Lint, A. L., Wakim, L. M., Heath, W. R.

and Carbone, F. R. (2003). Epidermal viral immunity induced by CD8alphaþ

dendritic cells but not by Langerhans cells. Science, 301, 1925–8.

Ardavın, C. (1997). Thymic dendritic cells. Immunol Today, 18, 350–61.

Ardavın, C. (2003). Origin, precursors and differentiation of mouse dendritic

cells. Nat. Rev. Immunol., 3, 582–90.

Ardavın, C., Wu, L., Li, C. L. and Shortman, K. (1993). Thymic dendritic cells and

T cells develop simultaneously in the thymus from a common precursor

population. Nature, 362, 761–3.

Asselin-Paturel, C., Brizard, G., Pin, J. J., Briere, F. and Trinchieri, G. (2003).

Mouse strain differences in plasmacytoid dendritic cell frequency and

function revealed by a novel monoclonal antibody. J. Immunol., 171,

6466–77.

Barchet, W., Cella, M., Odermatt, B., Asselin-Paturel, C., Colonna, M. and

Kalinke, U. (2002). Virus-induced interferon alpha production by a dendritic

cell subset in the absence of feedback signaling in vivo. J. Exp. Med., 195,

507–16.

Barchet, W., Cella, M. and Colonna, M. (2005). Plasmacytoid dendritic cells-virus

experts of innate immunity. Semin. Immunol., 17, 253–61.

Belz, G. T., Behrens, G. M., Smith, C. M., Miller, J. F., Jones, C., Lejon, K.,

Fathman, C. G., Mueller, S. N., Shortman, K., Carbone, F. R. and

Heath, W. R. (2002). The CD8alpha(þ) dendritic cell is responsible for

�19

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

inducing peripheral self-tolerance to tissue-associated antigens. J. Exp. Med.,

196, 1099–104.

Belz, G. T., Smith, C. M., Kleinert, L., Reading, P., Brooks, A., Shortman, K.,

Carbone, F. R. and Heath, W. R. (2004). Distinct migrating and

nonmigrating dendritic cell populations are involved in MHC

class I-restricted antigen presentation after lung infection with virus.

Proc. Natl Acad. Sci. U S A, 101, 8670–5.

Belz, G. T., Shortman, K., Bevan, M. J. and Heath, W. R. (2005). CD8alphaþ

dendritic cells selectively present MHC class I-restricted noncytolytic viral

and intracellular bacterial antigens in vivo. J. Immunol., 175, 196–200.

Benvenuti, F., Hugues, S., Walmsley, M., Ruf, S., Fetler, L., Popoff, M.,

Tybulewicz, V. L. and Amigorena, S. (2004). Requirement of Rac1 and Rac2

expression by mature dendritic cells for T cell priming. Science, 305, 1150–3.

Blasius, A., Vermi, W., Krug, A., Facchetti, F., Cella, M. and Colonna, M. (2004). A

cell-surface molecule selectively expressed on murine natural interferon-

producing cells that blocks secretion of interferon-alpha. Blood, 103, 4201–6.

Borkowski, T. A., Letterio, J. J., Farr, A. G. and Udey, M. C. (1996). A role for

endogenous transforming growth factor beta 1 in Langerhans cell biology:

the skin of transforming growth factor beta 1 null mice is devoid of

epidermal Langerhans cells. J. Exp. Med., 184, 2417–22.

Chicha, L., Jarrossay, D. and Manz, M. G. (2004). Clonal type I

interferon-producing and dendritic cell precursors are contained in

both human lymphoid and myeloid progenitor populations. J. Exp. Med.,

200, 1519–24.

Colonna, M., Trinchieri, G. and Liu, Y. J. (2004). Plasmacytoid dendritic cells in

immunity. Nat. Immunol., 5, 1219–26.

D’Amico, A. and Wu, L. (2003). The early progenitors of mouse dendritic

cells and plasmacytoid predendritic cells are within the bone marrow

hemopoietic precursors expressing Flt3. J. Exp. Med., 198, 293–303.

Daro, E., Pulendran, B., Brasel, K., Teepe, M., Pettit, D., Lynch, D. H., Vremec, D.,

Robb, L., Shortman, K., McKenna, H. J., Maliszewski, C. R. and

Maraskovsky, E. (2000). Polyethylene glycol-modified GM-CSF expands

CD11b(high)CD11c(high) but not CD11b(low)CD11c(high) murine

dendritic cells in vivo: a comparative analysis with Flt3 ligand. J. Immunol.,

165, 49–58.

De Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P.,

Urbain, J., Leo, O. and Moser, M. (1996). Regulation of dendritic cell

numbers and maturation by lipopolysaccharide in vivo. J. Exp. Med., 184,

1413–24.

�

20

C.

AR

DA

VIN

den Haan, J. M., Lehar, S. M. and Bevan, M. J. (2000). CD8(þ) but not CD8(�)

dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med., 192,

1685–96.

Edwards, A. D., Diebold, S. S., Slack, E. M., Tomizawa, H., Hemmi, H.,

Kaisho, T., Akira, S. and Reis e Sousa, C. (2003). Toll-like receptor

expression in murine DC subsets: lack of TLR7 expression by CD8 alphaþ

DC correlates with unresponsiveness to imidazoquinolines. Eur. J.

Immunol., 33, 827–33.

Fallarino, F., Grohmann, U., Vacca, C., Bianchi, R., Fioretti, M. C. and Puccetti, P.

(2002). CD40 ligand and CTLA-4 are reciprocally regulated in the Th1 cell

proliferative response sustained by CD8(þ) dendritic cells. J. Immunol., 169,

1182–8.

Filippi, C., Hugues, S., Cazareth, J., Julia, V., Glaichenhaus, N. and Ugolini, S.

(2003). CD4þ T cell polarization in mice is modulated by strain-specific

major histocompatibility complex-independent differences within dendritic

cells. J. Exp. Med., 198, 201–9.

Fleeton, M. N., Contractor, N., Leon, F., Wetzel, J. D., Dermody, T. S. and

Kelsall, B. L. (2004). Peyer’s patch dendritic cells process viral antigen

from apoptotic epithelial cells in the intestine of reovirus-infected mice.

J. Exp. Med., 200, 235–45.

Fujii, S., Shimizu, K., Smith, C., Bonifaz, L. and Steinman, R. M. (2003).

Activation of natural killer T cells by alpha-galactosylceramide rapidly

induces the full maturation of dendritic cells in vivo and thereby acts as an

adjuvant for combined CD4 and CD8 T cell immunity to a coadministered

protein. J. Exp. Med., 198, 267–79.

Gilliet, M., Boonstra, A., Paturel, C., Antonenko, S., Xu, X. L., Trinchieri, G.,

O’Garra, A. and Liu, Y. J. (2002). The development of murine plasmacytoid

dendritic cell precursors is differentially regulated by FLT3-ligand and

granulocyte/macrophage colony-stimulating factor. J. Exp. Med., 195, 953–8.

Hacker, C., Kirsch, R. D., Ju, X. S., Hieronymus, T., Gust, T. C., Kuhl, C.,

Jorgas, T., Kurz, S. M., Rose-John, S., Yokota, Y. and Zenke, M. (2003).

Transcriptional profiling identifies Id2 function in dendritic cell

development. Nat. Immunol., 4, 380–6.

Henri, S., Vremec, D., Kamath, A., Waithman, J., Williams, S., Benoist, C.,

Burnham, K., Saeland, S., Handman, E. and Shortman, K. (2001). The

dendritic cell populations of mouse lymph nodes. J. Immunol., 167, 741–8.

Honda, K., Mizutani, T. and Taniguchi, T. (2004). Negative regulation of

IFN-alpha/beta signaling by IFN regulatory factor 2 for homeostatic

development of dendritic cells. Proc. Natl Acad. Sci. U S A, 101, 2416–21.

�21

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

Ichikawa, E., Hida, S., Omatsu, Y., Shimoyama, S., Takahara, K., Miyagawa, S.,

Inaba, K. and Taki, S. (2004). Defective development of splenic and

epidermal CD4þ dendritic cells in mice deficient for IFN regulatory factor-2.

Proc. Natl Acad. Sci. U S A, 101, 3909–14.

Itano, A. A., McSorley, S. J., Reinhardt, R. L., Ehst, B. D., Ingulli, E.,

Rudensky, A. Y. and Jenkins, M. K. (2003). Distinct dendritic cell

populations sequentially present antigen to CD4 T cells and stimulate

different aspects of cell-mediated immunity. Immunity, 19, 47–57.

Iwasaki, A. (2003). The importance of CD11bþ dendritic cells in CD4þ T

cell activation in vivo: with help from interleukin 1. J. Exp. Med., 198, 185–90.

Iwasaki, A. and Kelsall, B. L. (1999). Freshly isolated Peyer’s patch, but not spleen,

dendritic cells produce interleukin 10 and induce the differentiation of T

helper type 2 cells. J. Exp. Med., 190, 229–39.

Iwasaki, A. and Kelsall, B. L. (2000). Localization of distinct Peyer’s patch

dendritic cell subsets and their recruitment by chemokines macrophage

inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid

organ chemokine. J. Exp. Med., 191, 1381–94.

Iwasaki, A. and Kelsall, B. L. (2001). Unique functions of CD11bþ, CD8 alphaþ,

and double-negative Peyer’s patch dendritic cells. J. Immunol., 166, 4884–90.

Iwasaki, A. and Medzhitov, R. (2004). Toll-like receptor control of the adaptive

immune responses. Nat. Immunol., 5, 987–95.

Iyoda, T., Shimoyama, S., Liu, K., Omatsu, Y., Akiyama, Y., Maeda, Y.,

Takahara, K., Steinman, R. M. and Inaba, K. (2002). The CD8þ dendritic

cell subset selectively endocytoses dying cells in culture and in vivo. J. Exp.

Med., 195, 1289–302.

Johansson, C. and Kelsall, B. L. (2005). Phenotype and function of intestinal

dendritic cells. Semin. Immunol., 17, 284–94.

Kabashima, K., Banks, T. A., Ansel, K. M., Lu, T. T., Ware, C. F. and

Cyster, J. G. (2005). Intrinsic lymphotoxin-beta receptor requirement

for homeostasis of lymphoid tissue dendritic cells. Immunity, 22, 439–50.

Kamogawa-Schifter, Y., Ohkawa, J., Namiki, S., Arai, N., Arai, K. and Liu, Y.

(2005). Ly49Q defines 2 pDC subsets in mice. Blood, 105, 2787–92.

Karsunky, H., Merad, M., Mende, I., Manz, M. G., Engleman, E. G.

and Weissman, I. L. (2005). Developmental origin of interferon-alpha-

producing dendritic cells from hematopoietic precursors. Exp. Hematol.,

33, 173–81.

Kobayashi, T., Walsh, P. T., Walsh, M. C., Speirs, K. M., Chiffoleau, E., King, C. G.,

Hancock, W. W., Caamano, J. H., Hunter, C. A., Scott, P., Turka, L. A. and

Choi, Y. (2003). TRAF6 is a critical factor for dendritic cell maturation and

development. Immunity, 19, 353–63.

�

22

C.

AR

DA

VIN

Krug, A., Veeraswamy, R., Pekosz, A., Kanagawa, O., Unanue, E. R., Colonna, M.

and Cella, M. (2003). Interferon-producing cells fail to induce proliferation of

naive T cells but can promote expansion and T helper 1 differentiation of

antigen-experienced unpolarized T cells. J. Exp. Med., 197, 899–906.

Krug, A., French, A. R., Barchet, W., Fischer, J. A., Dzionek, A., Pingel, J. T.,

Orihuela, M. M., Akira, S., Yokoyama, W. M. and Colonna, M. (2004).

TLR9-dependent recognition of MCMV by IPC and DC generates

coordinated cytokine responses that activate antiviral NK cell

function. Immunity, 21, 107–19.

Laouar, Y., Welte, T., Fu, X. Y. and Flavell, R. A. (2003). STAT3 is required for Flt3L-

dependent dendritic cell differentiation. Immunity, 19, 903–12.

Leenen, P. J., Radosevic, K., Voerman, J. S., Salomon, B., van Rooijen, N.,

Klatzmann, D. and van Ewijk, W. (1998). Heterogeneity of mouse

spleen dendritic cells: in vivo phagocytic activity, expression of

macrophage markers, and subpopulation turnover. J. Immunol., 160,

2166–73.

Leon, B., Lopez-Bravo, M. and Ardavın, C. (2005). Monocyte-derived dendritic

cells. Semin. Immunol., 17, 313–18.

Liu, K., Iyoda, T., Saternus, M., Kimura, Y., Inaba, K. and Steinman, R. M. (2002).

Immune tolerance after delivery of dying cells to dendritic cells in situ.

J. Exp. Med., 196, 1091–7.

Maldonado-Lopez, R., Maliszewski, C., Urbain, J. and Moser, M. (2001).

Cytokines regulate the capacity of CD8alpha(þ) and CD8alpha(�)

dendritic cells to prime Th1/Th2 cells in vivo. J. Immunol., 167, 4345–50.

Manz, M. G., Traver, D., Miyamoto, T., Weissman, I. L. and Akashi, K. (2001).

Dendritic cell potentials of early lymphoid and myeloid progenitors.

Blood, 97, 3333–41.

Martin, P., Ruiz, S. R., del Hoyo, G. M., Anjuere, F., Vargas, H. H.,

Lopez-Bravo, M. and Ardavın, C. (2002). Dramatic increase in lymph

node dendritic cell number during infection by the mouse mammary

tumor virus occurs by a CD62L-dependent blood-borne DC recruitment.

Blood, 99, 1282–8.

Mayerova, D., Parke, E. A., Bursch, L. S., Odumade, O. A. and Hogquist, K. A.

(2004). Langerhans cells activate naive self-antigen-specific CD8 Tcells in the

steady state. Immunity, 21, 391–400.

McKenna, H. J., Stocking, K. L., Miller, R. E., Brasel, K., De Smedt, T.,

Maraskovsky, E., Maliszewski, C. R., Lynch, D. H., Smith, J., Pulendran, B.,

Roux, E. R., Teepe, M., Lyman, S. D. and Peschon, J. J. (2000). Mice

lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic

progenitor cells, dendritic cells, and natural killer cells. Blood, 95, 3489–97.

�23

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

Pooley, J. L., Heath, W. R. and Shortman, K. (2001). Cutting edge: intravenous

soluble antigen is presented to CD4 T cells by CD8-dendritic cells,

but cross-presented to CD8 T cells by CD8þ dendritic cells. J. Immunol.,

166, 5327–30.

Prakash, A., Smith, E., Lee, C. K. and Levy, D. E. (2005). Tissue-specific positive

feedback requirements for production of type I interferon following virus

infection. J. Biol. Chem., 280, 18651–7.

Reis e Sousa, C., Hieny, S., Scharton-Kersten, T., Jankovic, D., Charest, H.,

Germain, R. N. and Sher, A. (1997). In vivo microbial stimulation

induces rapid CD40 ligand-independent production of interleukin 12

by dendritic cells and their redistribution to T cell areas. J. Exp. Med., 186,

1819–29.

Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R.,

Granucci, F., Kraehenbuhl, J. P. and Ricciardi-Castagnoli, P. (2001).

Dendritic cells express tight junction proteins and penetrate gut epithelial

monolayers to sample bacteria. Nat. Immunol., 2, 361–7.

Ritter, U., Meissner, A., Scheidig, C. and Korner, H. (2004). CD8 alpha- and

Langerin-negative dendritic cells, but not Langerhans cells, act as principal

antigen-presenting cells in leishmaniasis. Eur. J. Immunol., 34, 1542–50.

Sato, A., Hashiguchi, M., Toda, E., Iwasaki, A., Hachimura, S. and

Kaminogawa, S. (2003). CD11bþ Peyer’s patch dendritic cells secrete

IL-6 and induce IgA secretion from naive B cells. J. Immunol., 171,

3684–90.

Scheinecker, C., McHugh, R., Shevach, E. M. and Germain, R. N. (2002).

Constitutive presentation of a natural tissue autoantigen exclusively by

dendritic cells in the draining lymph node. J. Exp. Med., 196, 1079–90.

Schiavoni, G., Mattei, F., Sestili, P., Borghi, P., Venditti, M., Morse, H. C., 3rd,

Belardelli, F. and Gabriele, L. (2002). ICSBP is essential for the development

of mouse type I interferon-producing cells and for the generation and

activation of CD8alpha(þ) dendritic cells. J. Exp. Med., 196, 1415–25.

Schiavoni, G., Mattei, F., Borghi, P., Sestili, P., Venditti, M., Morse, H. C., 3rd,

Belardelli, F. and Gabriele, L. (2004). ICSBP is critically involved in the

normal development and trafficking of Langerhans cells and dermal

dendritic cells. Blood, 103, 2221–8.

Schlecht, G., Garcia, S., Escriou, N., Freitas, A. A., Leclerc, C. and Dadaglio, G.

(2004). Murine plasmacytoid dendritic cells induce effector/memory CD8þ

T-cell responses in vivo after viral stimulation. Blood, 104, 1808–15.

Schulz, O. and Reis e Sousa, C. (2002). Cross-presentation of cell-associated

antigens by CD8alphaþ dendritic cells is attributable to their ability to

internalize dead cells. Immunology, 107, 183–9.

�

24

C.

AR

DA

VIN

Shigematsu, H., Reizis, B., Iwasaki, H., Mizuno, S., Hu, D., Traver, D., Leder, P.,

Sakaguchi, N. and Akashi, K. (2004). Plasmacytoid dendritic cells activate

lymphoid-specific genetic programs irrespective of their cellular origin.

Immunity, 21, 43–53.

Shortman, K. and Liu, Y. J. (2002). Mouse and human dendritic cell subtypes.

Nat. Rev. Immunol., 2, 151–61.

Steinman, R. M. and Nussenzweig, M. C. (2002). Avoiding horror autotoxicus:

the importance of dendritic cells in peripheral T cell tolerance. Proc. Natl

Acad. Sci. U SA, 99, 351–8.

Suzuki, S., Honma, K., Matsuyama, T., Suzuki, K., Toriyama, K., Akitoyo, I.,

Yamamoto, K., Suematsu, T., Nakamura, M., Yui, K. and Kumatori, A.

(2004). Critical roles of interferon regulatory factor 4 in CD11bhigh

CD8alpha� dendritic cell development. Proc. Natl Acad. Sci. U SA, 101,

8981–6.

Tamura, T., Tailor, P., Yamaoka, K., Kong, H. J., Tsujimura, H., O’Shea, J. J.,

Singh, H. and Ozato, K. (2005). IFN regulatory factor-4 and -8 govern

dendritic cell subset development and their functional diversity. J. Immunol.,

174, 2573–81.

Thery, C., Duban, L., Segura, E., Veron, P., Lantz, O. and Amigorena, S. (2002).

Indirect activation of naive CD4þ T cells by dendritic cell-derived exosomes.

Nat. Immunol., 3, 1156–62.

Traver, D., Akashi, K., Manz, M., Merad, M., Miyamoto, T., Engleman, E. G.

and Weissman, I. L. (2000). Development of CD8alpha-positive

dendritic cells from a common myeloid progenitor. Science, 290,

2152–4.

Tsujimura, H., Tamura, T. and Ozato, K. (2003). Cutting edge: IFN

consensus sequence binding protein/IFN regulatory factor 8 drives the

development of type I IFN-producing plasmacytoid dendritic cells.

J. Immunol., 170, 1131–5.

Turley, S., Poirot, L., Hattori, M., Benoist, C. and Mathis, D. (2003). Physiological

beta cell death triggers priming of self-reactive T cells by dendritic cells in

a type-1 diabetes model. J. Exp. Med., 198, 1527–37.

Valladeau, J. and Saeland, S. (2005). Cutaneous dendritic cells. Semin.

Immunol., 17, 273–83.

Villadangos, J. A. and Heath, W. R. (2005). Life cycle, migration and

antigen presenting functions of spleen and lymph node dendritic

cells: Limitations of the Langerhans cells paradigm. Semin. Immunol.,

17, 262–72.

Wu, L., Nichogiannopoulou, A., Shortman, K. and Georgopoulos, K. (1997).

Cell-autonomous defects in dendritic cell populations of Ikaros mutant

�25

SU

BP

OP

UL

AT

ION

SA

ND

DIF

FE

RE

NT

IAT

ION

OF

MO

US

ED

CS

mice point to a developmental relationship with the lymphoid lineage.

Immunity, 7, 483–92.

Wu, L., D’Amico, A., Winkel, K. D., Suter, M., Lo, D. and Shortman, K.

(1998). RelB is essential for the development of myeloid-related

CD8alpha� dendritic cells but not of lymphoid-related CD8alphaþ

dendritic cells. Immunity, 9, 839–47.

Wu, L., D’Amico, A., Hochrein, H., O’Keeffe, M., Shortman, K. and Lucas, K.

(2001). Development of thymic and splenic dendritic cell populations

from different hemopoietic precursors. Blood, 98, 3376–82.

Zhang, Y., Zhang, Y. Y., Ogata, M., Chen, P., Harada, A., Hashimoto, S. and

Matsushima, K. (1999). Transforming growth factor-beta1 polarizes

murine hematopoietic progenitor cells to generate Langerhans cell-like

dendritic cells through a monocyte/macrophage differentiation pathway.

Blood, 93, 1208–20.

�

26

C.

AR

DA

VIN

CHAPTER 2

Toll-like receptor signaling

Osamu Takeuchi and Shizuo AkiraOsaka University

2.1 INTRODUCTION

Toll-like receptors (TLRs) play essential roles in innate immune responses1,2.

The name TLR is derived from a Drosophila protein, Toll, which detects

fungal infection in the fruit fly. The immune system in Drosophila is entirely

dependent on a limited number of germline-encoded receptors for pathogen

recognition. In contrast, the vertebrate immune system is characterized by

the evolution of acquired immunity in addition to innate immunity.

Acquired immunity is mediated by T and B cells, which utilize rearranged

receptors. This system is advantageous for detecting pathogens with high

specificity, eradicating infection in the late stages and establishing an

immunological memory. However, the mammalian innate immune

system plays critical roles in the initial defense against invading pathogens

and subsequent activation of the acquired immune system. Innate immune

cells, such as macrophages and dendritic cells (DCs), sense pathogens

through TLRs, phagocytose them and evoke immune responses.

To date, 12 different TLRs have been reported in either humans or mice.

The innate immunity system targets a set of molecular structures that are

unique to microorganisms and shared by various pathogens, but absent from

host cells. By recognizing these ‘‘pathogen-specific’’ patterns, the innate

immunity system is able to prevent autoimmune responses. Members of

the TLR family of proteins are characterized by extracellular leucine-rich

repeat (LRR) motifs responsible for ligand recognition, a transmembrane

region and a cytoplasmic tail containing a Toll/IL-1 receptor homology

(TIR) domain. Extensive studies have identified the pathogenic components

recognized by each TLR. TLR1, 2, 4, 5, 6 and 11 are involved in the

recognition of microbial components that contain lipids, sugars and

proteins. In detail, lipopolysaccharide (LPS) is recognized by TLR43,4,

bacterial di-acyl lipoprotein is recognized by a heterodimer of TLR2 and

TLR65,6, tri-acyl lipoprotein is recognized by a heterodimer of TLR1 and

TLR27,8, bacterial flagellin is recognized by TLR59 and uropathogenic

bacteria and a profilin-like molecule from Toxoplasma gondii are detected

by TLR1110,11. In contrast, TLR3, 7, 8 and 9 recognize nucleotides from

pathogens. For example, double-stranded RNA (dsRNA) is recognized by

TLR312. viral GU-rich single-stranded RNA (ssRNA) is recognized by TLR7

in mice and TLR8 in humans13,14 and bacterial or viral unmethylated DNA

with CpG motifs is recognized by TLR915–17. TLR1, 2, 4, 5, 6 and 11 are

expressed on the plasma membrane, whereas the TLRs that recognize

nucleotides are localized to intracellular endosomes or lysosomes18.

2.2 TLR SIGNALING AND TIR DOMAIN-CONTAINING ADAPTOR

MOLECULES

Upon recognition of pathogenic components and ligand stimulation, TLRs

activate similar, but distinct, signaling pathways via their TIR domains.

These intracellular signaling pathways are responsible for various cellular

and systemic immune responses, and have pleiotrophic effects including

the production of proinflammatory cytokines, chemokines and interferons

(IFNs), upregulation of surface co-stimulation molecule expression, phago-

cytosis of bacteria and induction of B cell proliferation. DCs play pivotal roles

in T cell activation and differentiation.

The initial step of TLR signaling is the recruitment of cytoplasmic

adaptor molecules to the receptor. These adaptors possess a TIR domain and

associate with the TIR domain of TLR/IL-1R via a homophilic interaction.

To date, five TIR domain-containing adaptor molecules have been identi-

fied in mammals, including myeloid differentiation factor 88 (MyD88),

TIR domain-containing adaptor inducing IFNb (TRIF), TIR domain-

containing adaptor protein (TIRAP)/MyD88 adaptor-like (MAL), TRIF-

related adaptor molecule (TRAM) and sterile a and HEAT-Armadillo

motifs (SARM). Four of these adaptors (MyD88, TRIF, TIRAP/Mal and

TRAM) are known to be involved in TLR signaling in mammals (Figure 2.1).

In contrast, a role for mammalian SARM has not yet been identified,

although the Caenorhabditis elegans SARM homologue has been implicated

in host defense against bacteria19,20. A distinct set of TIR domain-containing

adaptors is recruited to each TLR, and a distinct response is evoked, at least

in part, by the combination of adaptors recruited. Among the adaptor

molecules, MyD88 and TRIF regulate the activation of the two main

signaling pathways leading to the production of proinflammatory cytokines

and type I IFNs, respectively. In addition, cell type-specific recruitment of

�

28

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

Fig

ure

2.1

.S

chem

eo

fth

eT

LR

-sig

nal

ing

pat

hw

ays.

(a)

MyD

88

-dep

end

ent

sig

nal

ing

pat

hw

ays.

Th

esi

gn

alin

gp

ath

way

sem

anat

ing

fro

mM

yD88

acti

vate

tran

scri

pti

on

fact

ors

resp

on

sib

lefo

rth

ein

du

ctio

no

fp

roin

flam

mat

ory

cyto

kin

eg

enes

.Th

ere

cru

itm

ento

fM

yD88

tore

cep

tors

ind

uce

sse

qu

enti

alac

tiva

tio

no

fIR

AK

s,

TR

AF6

,TA

K1

,th

eIK

Kco

mp

lex

and

NF

-kB

.TA

K1

ph

osp

ho

ryla

tes

MK

Ks,

wh

ich

intu

rnac

tiva

teM

AP

kin

ases

such

asJN

Kan

dp38,t

her

eby

ind

uci

ng

AP

-1ac

tiva

tio

n.

IRF

-5as

soci

ates

wit

hT

RA

F6

and

MyD

88

,an

dtr

ansl

oca

tes

toth

en

ucl

eus

inre

spo

nse

tost

imu

lati

on

.(b

)T

RIF

-dep

end

ent

sig

nal

ing

pat

hw

ays.

IncD

Cs,

the

TR

IF-

dep

end

ent

sig

nal

ing

pat

hw

ays

eman

atin

gfr

om

TL

R3

and

TL

R4

lead

toth

ein

du

ctio

no

fb

oth

pro

infl

amm

ato

rycy

tok

ines

and

typ

eI

IFN

s.T

RIF

inte

ract

sw

ith

TR

AF6,

RIP1

and

TB

K1.

TR

AF6

and

RIP1

acti

vate

NF

-kB

,w

her

eas

TB

K1

and

IKK

-ip

ho

sph

ory

late

IRF

-3an

dIR

F-7

,th

ereb

yle

adin

gto

the

ind

uct

ion

of

typ

eI

IFN

gen

es.

(c)

Mec

han

ism

so

fty

pe

IIF

Nin

du

ctio

nin

pD

Cs.

TL

R7

and

TL

R9

recr

uit

aco

mp

lex

of

MyD

88

,IR

AK1,

IRA

K4,

TR

AF6

and

IRF

-7in

pD

Cs.

Sti

mu

lati

on

wit

h

Cp

G-D

NA

resu

lts

inth

ep

ho

sph

ory

lati

on

and

nu

clea

rtr

ansl

oca

tio

no

fIR

F-7

,fo

llo

wed

by

the

exp

ress

ion

of

IFN

-ag

enes

.T

LR7

and

TL

R9

stim

ula

tio

nal

soac

tiva

tes

NF

-kB

thro

ug

hIR

AK4

ina

TR

AF6

-dep

end

ent

man

ner

.

signaling machinery to each receptor is also important for the adaptive

response to TLR ligands. In the following sections, we focus on the

mechanisms for how TLRs stimulate distinct signaling pathways leading

to adaptive responses.

2.3 MYD88-DEPENDENT SIGNALING: A COMMON PATHWAY

FOR MOST TLRS FOR THE INDUCTION OF

PROINFLAMMATORY CYTOKINES

MyD88 was the first adaptor molecule identified for TLR/IL-1R signal-

ing21,22. The MyD88-dependent signaling pathways are shared by all the

known TLRs, except for TLR3. MyD88 is comprised of an N-terminal death

domain (DD) and a C-terminal TIR domain, and is recruited to the TIR

domain of TLR/IL-1R in response to ligand stimulation. Analysis of MyD88-

deficient (MyD88�/�) mice revealed an essential role for this adaptor in

IL-1R/TLR signaling23–25. since TLR2-, 5-, 7- and 9-mediated activation of

nuclear factor (NF)-kB was abrogated in these mice. Another of the adaptor

molecules, TIRAP/Mal, is also involved in MyD88-dependent signaling26–29.

TIRAP/Mal is specifically responsible for TLR2 and TLR4 signaling, and has

been suggested to play a role in bridging TLR2 and TLR4 to MyD88.

Upon recruitment to a TLR, MyD88 interacts with IL-1R-associated

kinases (IRAKs), including IRAK1 and IRAK4 (Figure 2.1). IRAKs are

composed of an N-terminal DD and an adjacent serine/threonine kinase

domain. Interaction of IRAK1 with IRAK4 induces IRAK1 phosphorylation

and activation30. IL-1R- and TLR-mediated signaling as well as cytokine

production were abrogated in IRAK4�/� mice31. Although IRAK1�/� mice

were reported to show impaired cytokine production in response to IL-1

and LPS stimulation, the mice were still capable of producing significant

amounts of cytokines32,33. This may be due to compensation by other IRAK

family members. The hyperphosphorylated IRAK1 recruits TRAF6 to the

receptor complex34. TRAF6 contains an N-terminal RING domain and a

conserved C-terminal TRAF domain. RING finger domains are also found in

the large family of E3 ubiquitin ligases, and TRAF6 was recently shown to

function as a ubiquitin ligase, together with an E2 ligase complex consisting

of Ubc13 and Uev1A, and catalyze the formation of a lysine-63 (K63)-linked

polyubiquitin chain of TRAF6 and NFkB essential modulator (NEMO)/IkB

kinase (IKK)g35. Ubiquitinated TRAF6 activates a complex of transforming

growth factor (TGF)b-activating kinase 1 (TAK1) and its associated proteins,

tubulin antisense-binding protein (TAB)1, TAB2 and TAB3. TAK1 belongs

�

30

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

to the mitogen-activated protein kinase kinase kinase (MAPKKK) family,

and has been reported to be involved in the IL-1b signaling pathway36.

Ubiquitin-dependent activation of TAK1 results in the phosphorylation

of IKKb, thereby leading to NF-kB activation. Knockdown of TAK1 by small

interfering RNA (siRNA) abolished the IKK activation induced by TNFaand IL-1b stimulation. TAB1, TAB2 and TAB3 were identified as adaptor

proteins that associate with TAK137,38. TAB2 and TAB3 bind preferentially to

K63-linked polyubiquitin chains through their zinc finger domains.

However, IL-1b-mediated activation of NF-kB and MAP kinases was not

impaired in mouse embryonic fibroblasts (MEFs) from TAB2�/� mice39.

Therefore, TAB2 and TAB3 may compensate for each other’s functions

in vivo. The activated IkB kinase (IKK) complex, composed of IKKa, IKKband NEMO/IKKg, phosphorylates IkB40. In resting cells, IkB forms a

complex with NF-kB, thereby sequestering NF-kB in the cytoplasm and

preventing its activation. Phosphorylated IkB is recognized and modified by

K48-linked polyubiquitin by an E3-ubiquitin ligase called bTrCP, resulting in

its proteasome-mediated degradation. Following degradation of IkB, the

released NF-kB translocates to the nucleus where it binds to promoter

regions containing kB sites to induce the expression of proinflammatory

cytokine genes.

In addition to NF-kB, MyD88-dependent signaling also activates MAP

kinases, including c-Jun N-terminal kinase (JNK) and p38. In turn, the

activated MAP kinases phosphorylate and activate AP-1 to induce the

expression of proinflammatory cytokine genes in cooperation with NF-kB.

The signaling pathway leading to MAP kinase activation is less well

understood. TAK1 is capable of phosphorylating MAP kinase kinase

(MKK) 6 in IL-1 signaling36. JNK and p38 are activated by the upstream

MKK3 and MKK6, respectively. A small G protein, Ras, has also been

implicated in IL-1-mediated activation of MKK3 and MKK641. Recently,

another MAPKKK, apoptosis signal-regulating kinase 1 (ASK1), was

reported to be responsible for p38 activation in response to LPS, but not

TLR2 or other ligands42. ASK1�/� mice showed impaired cytokine responses

against LPS. Furthermore, ASK1 can interact with TRAF6, and LPS-induced

production of intracellular reactive oxygen species was impaired in ASK1�/�

cells.

In addition to NF-kB and MAP kinases, a transcription factor known as

IFN-regulatory factor (IRF) 5 is involved in the expression of proinflamma-

tory cytokines. IRF-5 interacts with MyD88 and TRAF6, and ligand

stimulation induces its translocation to the nucleus where it activates

�31

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

cytokine gene transcription43. DCs from IRF-5�/� mice showed impaired

productions of IL-6, TNF-a and IL-12 in response to various TLR stimuli.

Although an in vitro study found that IRF-5 was involved in TLR7-mediated

type I IFN production44, it has also been reported to be dispensable for

TLR-induced IFN production43.

TLR signaling is negatively regulated by various molecules, including

several transmembrane receptors. T1/ST2 belongs to the IL-1R family

and has been suggested as a molecule responsible for Th2 development.

In addition, T1/ST2 suppresses IL-1R and TLR4 signaling by sequestering

MyD88 and TIRAP/Mal45. RP105, a LRR-containing membrane protein, has

been implicated in the negative regulation of TLR4 signaling46. However,

this protein functions as a positive regulator of LPS signaling in B cells47.

Triad3A, a RING finger protein, functions as a ubiquitin ligase and catalyzes

the ubiquitination and degradation of TLR4 and TLR948. Knockdown of

Triad3A by siRNA enhanced the responses to TLR ligands. In addition,

DAP12, an adaptor protein containing an immunoreceptor tyrosine-based

activation motif (ITAM) for receptor signaling on natural killer cells, also

negatively regulates TLR signaling49. DAP12�/� mice showed increased

production of cytokines, enhanced susceptibility to LPS-induced shock and

enhanced resistance to infection by Listeria monocytogenes49. Intracellular

signaling molecules, including MyD88, IRAKs and TRAF6, are also

modulated and negatively regulated to prevent aberrant activation of

inflammatory responses. A splicing variant of MyD88, MyD88(S), lacking

the intermediate region, behaves as a dominant-negative regulator of IL-1R

and TLR4 signaling50. Among the four IRAK family members, IRAK-M has

been reported to function as a negative regulator. Tollip, a small protein with

an internal C2 domain and a C-terminal CUE domain, has also been

implicated in IL-1R/TLR signaling51. Tollip associates with IRAK1, and its

overexpression perturbs IL-1R-, TLR2- and TLR4-mediated signaling path-

ways, thereby suggesting that it acts as a negative regulator of TLR

signaling52. A20, an enzyme that modifies ubiquitination of TRAF6, is

required for the termination of TLR-induced NF-kB activation53.

2.4 TRIF-DEPENDENT PATHWAY: CONNECTING TLR SIGNALING

TO TYPE I IFNS

TRIF is another TIR domain-containing adaptor molecule that is responsible

for TLR3 and TLR4 signaling (Figure 2.1b)54,55. TRIF associates with TLR3

or another of the adaptors, TRAM, via their TIR domains. Since TLR3 and

TLR4 can recognize viral components, a feature of TRIF is its ability to

�

32

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

induce type I IFNs. Generation of TRIF�/� mice and studies on a mouse

strain harboring a mutation in TRIF revealed that TRIF is essential for the

responses induced by TLR3 and TLR4 ligands. TRIF�/� mice showed

defective production of proinflammatory cytokines and expression of IFN-

inducible genes in response to LPS or poly I:C. Furthermore, LPS-mediated

activation of IRF-3 was abrogated in TRIF�/� macrophages.

Recent studies have revealed that two IKK-related kinases, namely

inducible IkB kinase (IKK-i; also known as IKKe) and TRAF-family member-

associated NF-kB activator (TANK)-binding kinase 1 (TBK1; also known as

T2K), are involved in IFN production upon TLR stimulation and viral

infection56,57. IKK-i and TBK1 can directly phosphorylate interferon

regulatory factor 3 (IRF-3) and IRF-7 in vitro and activate the IFNbpromoter56. TBK1�/�, but not IKK-i�/�, MEFs showed severely impaired

induction of IFNb and IFN-inducible genes in response to LPS, intracellular

introduction of poly I:C and RNA virus infection. Activation of IRF-3, but not

NF-kB, in response to LPS and poly(I:C) was also diminished in TBK1�/�

cells. IKK-i/TBK1 double-deficient MEFs failed to express any detectable

levels of IFNb and IFN-inducible genes in response to poly I:C, indicating

that both IKK-i and TBK1 contribute to the IFN pathway58–60.

The transcription factors IRF-3 and IRF-7 are essential for the expression

of type I IFNs in response to viral infection and TLR stimulation. Following

phosphorylation by TBK1/IKK-i, IRF-3 and IRF-7 form homodimers or

heterodimers and translocate into the nucleus. Once there, they interact

with the coactivator proteins CBP and p300 and subsequently bind to

IFN-stimulated regulatory elements (ISREs) present in the promoters of a

set of IFN-inducible genes to induce their expression61. Secreted type I IFNs,

in turn, activate their receptors in both autocrine and paracrine manners.

This activates IFN-stimulated gene factor 3 (ISGF3), which consists of signal

transducer and activator of transcription (STAT) 1, STAT2 and p40/IRF-9,

thereby amplifying the response. IRF-3�/� mice showed defective induction

of IFNb in response to LPS, indicating a critical role for IRF-3 in the TLR4-

induced IFN response62. IRF-7�/� mice showed profound defects in the

induction of type I IFNs, but not in the activation of NF-kB, in response to

CpG-DNA and viral infection, and therefore succumbed to infection with

several viruses63. Furthermore, cells from IRF-3/IRF-7 double-deficient mice

showed completely abrogated production of type I IFNs upon exposure to

several viruses, suggesting that both IRF-3 and IRF-7 are involved in the

initial production of IFNs in response to viral exposure63.

TRIF-dependent signaling also activates NF-kB. TRIF interacts with

TRAF6 through TRAF6 binding motifs in the N-terminal portion of TRIF,

�33

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

and with receptor interacting protein (RIP1 and RIP3) via a C-terminal RIP

homotypic interaction motif (RHIM)64,65. Poly I:C-induced NF-kB activation

was abolished in RIP1�/� MEFs. In addition, overexpression of TRIF was

found to induce apoptosis in some cells66. Deletion of the C-terminal RHIM

domain abolished the activity67.

TLR4-mediated NF-kB activation is regulated by either MyD88 or TRIF.

In TLR4-signaling, MyD88 regulates rapid activation of NF-kB, while TRIF is

responsible for sustained activation. Mice lacking both MyD88 and TRIF

were unable to activate NF-kB or MAP kinases as well as IRFs, indicating that

these two adaptors are responsible for TLR4 signaling68. Microarray analyses

of LPS-inducible genes in MyD88�/�, TRIF�/� and MyD88�/� TRIF�/�

macrophages revealed that these two adaptors are essential for the induction

of all genes in response to LPS stimulation69. On the other hand, cells lacking

either MyD88 or TRIF could still induce various genes, with the exception of

proinflammatory cytokines and IFN-inducible genes. These findings suggest

that many NF-kB target genes are ambiguously regulated by MyD88 or TRIF

whose signaling governs early or late activation of NF-kB69.

TRAM, another TIR domain-containing adaptor, is specifically involved

in TLR4, but not TLR3, signaling70,71. Overexpression of TRAM activates

IRF-3 and NF-kB-dependent gene induction. LPS-induced cytokine produc-

tion and IFN-inducible gene expression were severely impaired in TRAM�/�

cells. Furthermore, TRAM�/� cells failed to sustain LPS-induced NF-kB

activation compared to wild-type cells71. In contrast, there were no defects in

the responses to poly I:C and CpG-DNA. TRAM can interact physically with

both TLR4 and TRIF, but no direct association was detected between TLR4

and TRIF. Taken together, these observations suggest that TRAM functions

to bridge TLR4 and TRIF, resulting in activation of the downstream signaling

pathway.

2.5 TLR7, 9 SIGNALING IN PLASMACYTOID DCS INDUCES

PRODUCTION OF TYPE I IFNS

DCs are subcategorized into conventional and plasmacytoid DCs by their

expressions of different surface molecules as well as their abilities to produce

cytokines and type I IFNs upon exposure to pathogens. Conventional DCs

(cDCs) express various TLRs, including TLR3, and stimulation with

TLR ligands results in the production of proinflammatory cytokines.

Stimulation of cDCs with poly I:C or LPS induces the expression of IFNband IFN-inducible genes, indicating that the TRIF-dependent pathway

mainly functions to induce type I IFNs in response to TLR stimulation.

�

34

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

A subset of DCs, plasmacytoid DCs (pDCs), expresses high levels of

TLR7 and TLR9, but not TLR3. pDCs produce large amounts of IFNa upon

exposure to viruses72. Ligand stimulation of TLR7 and TLR9 results in the

production of IFNa. Among the TLR9 ligands, A/D-type CpG-DNAs, but not

conventional B/K-type CpG-DNAs, are potent inducers of IFNa, although the

production of proinflammatory cytokines is vigorously induced by both types

of CpG-DNAs73. In addition, infection with several DNA and RNA viruses

also induces IFN-a production by pDCs in a TLR7- or TLR9-dependent

manner14,16,74. Interestingly, the type I IFN induction in pDCs is indepen-

dent of TBK1. In pDCs, MyD88 was found to directly associate with IRF-7,

which then undergoes modifications such as phosphorylation and ubiqui-

tination (Figure 2.1c)75,76. IRAK4, IRAK1 and TRAF6are also recruited to the

complex. MyD88�/� and IRAK4�/� pDCs failed to activate both NF-kB

and IRF-7 in response to TLR9 ligands. In contrast, IRAK-1 plays a key role

in the expression of type I IFNs, but not proinflammatory cytokines77.

In IRAK1�/� pDCs, TLR9-mediated nuclear translocation of IRF-7 was

specifically abrogated. In addition, IRAK1 can phosphorylate IRF-7 in vitro,

suggesting that IRAK1 possibly functions as an IRF-7 kinase. The activated

IRF-7 induces the expression of IFNa genes.

The mechanisms of the cell type-specific IFNa production in response

to A/D-type CpG-DNAs are intriguing to explore. A/D-type CpG-DNAs

are structurally different from conventional CpG-DNAs, since they possess

a phosphorothioate-modified poly G stretch at the 5’ and 3’ ends and a

phosphodiester CpG motif in the central portion. It has been hypothesized

that pDCs, but not cDCs, constitutively express IRF-7, thereby explaining

the induction mechanism for type I IFNs78. Recently, it was proposed that

A/D-type CpG-DNAs are retained in endosomal vesicles in pDCs for a long

time, thus facilitating encounters between the DNA and TLR9-MyD88-IRF-7

complexes79. In cDCs, A/D-type CpG-DNAs are rapidly moved to lysosomal

vesicles. B/K-type CpG-DNAs, which induce lower levels of IFNa production,

acquire this activity after treatment with a cationic lipid that maintains

the DNA in endosomes in pDCs. However, this study is controversial to

several previous findings. First, although A/D-type CpG-DNA-induced IFNaproduction is specific to pDCs, the production of proinflammatory cytokines,

including TNF-a, IL-6 and IL-12, is vigorously induced in response to both

A/D-type and B/K-type CpG-DNAs in cDCs. These proinflammatory

cytokines are also induced in a TLR9-MyD88-dependent fashion, suggesting

that MyD88-dependent signaling is not impaired in cDCs. Moreover,

low concentrations of B/K-type CpG-DNAs are known to induce IFNa in

pDCs, and this IFNa-inducing activity is lost at higher concentrations73.

�35

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

These findings imply that the differences between A/D-type and B/K-type

DNAs, and between pDCs and cDCs, cannot be fully explained by endosomal

retention of CpG-DNAs alone. Future studies are therefore necessary

to elucidate the additional mechanisms of the type I IFN production in

response to TLR7 and TLR9 ligands in pDCs.

2.6 INTRACELLULAR RECOGNITION OF PATHOGENS

TLRs recognize pathogens at either the cell surface or lysosome/endosome

membranes, indicating that this system is invalid for the detection of

pathogens that have invaded the cytosol. Recent studies have revealed that

such pathogens already present in the cytoplasm are detected by various

cytoplasmic receptors without a transmembrane domain.

Synthesis of dsRNA in the infected cells is required for the replication

of ssRNA viruses, and such viral dsRNA is believed to be a virus-specific

molecular structure recognized by hosts. Most virus-infected cells produce

type I IFNs in a TLR3-independent manner, implying that the mechanism

for dsRNA recognition in the cytoplasm plays an important role in the IFN

response. Protein kinase R (PKR) has been implicated in viral dsRNA

recognition. PKR belongs to a family of proteins containing dsRNA-binding

domains, and is induced in response to stimulation with IFNs. Activation of

PKR results in growth arrest of the cells via eIF2a phosphorylation. Several

reports have demonstrated that virus-induced type I IFN production

is modestly impaired in PKR�/� mice80. Furthermore, PKR�/� mice

showed defective induction of apoptosis in response to poly I:C and

bacterial burdens. However, PKR�/� mice have also been reported to show

no alterations in their IFN responses to viral exposure81.

Recently, retinoic acid-inducible protein-I (RIG-I), an IFN-inducible

protein containing caspase recruitment domains (CARDs) and a helicase

domain, has been identified as a cytoplasmic dsRNA detector. RIG-I also

interacts with poly I:C82. RIG-I overexpression conferred Newcastle Disease

virus (NDV)- and dsRNA-mediated IFN responses on the cells. Mda-5,

a molecule showing homology to RIG-I, has also been implicated in the

recognition of viral dsRNA83. RIG-I�/� mice generally showed embryonic

lethality due to liver degeneration. RIG-I�/� MEFs did not produce

IFNb, activate IFN-inducible genes or induce proinflammatory cytokines

in response to infections with NDV, Sendai virus and Vesicular Stomatitis

virus (VSV)84. NDV-induced activation of NF-kB and ISRE-containing

genes was abrogated in RIG-I�/� cells. RIG-I overexpression in TBK1/IKK-i

double-deficient cells failed to activate the IFNb promoter, indicating that

�

36

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

RIG-I acts upstream of TBK1/IKK-i and governs both NF-kB and IRFs.

Furthermore, cDCs derived from the few RIG-I�/� mice born alive also failed

to induce type I IFNs and cytokines upon exposure to NDV. Interestingly,

pDCs from RIG-I�/� mice produced a comparable amount of IFNa to pDCs

from wild-type mice upon NDV stimulation, indicating that cDCs and pDCs

mainly utilize different mechanisms for the induction of IFNs84.

In MEFs, FADD and RIP1 are required for type I IFN responses to

stimulation with dsRNA, and FADD�/� or RIP1�/� MEFs were highly

susceptible to VSV infection85. FADD and RIP1 act upstream of TBK1/IKK-i,

suggesting that these molecules transduce signals from RIG-I to TBK1.

However, the cited report85 only described the response to poly I:C,

and not that against viral infection. Further analyses are therefore required

to clarify the roles of FADD and RIP1 in the IFN response to viral infection.

The mechanism of RIG-I-induced signaling as well as the relationships

among RIG-I, Mda-5 and TLR signaling will be extremely interesting to

explore.

CARD-containing proteins are also involved in recognizing bacteria in

the cytoplasm. These proteins include nucleotide-binding oligomerization

domain (NOD1 and NOD2), which both contain N-terminal CARD domains.

NOD1 detects g-d-glutamyl-meso diaminopimelic acid (iE-DAP) found in

Gram-negative bacterial peptidoglycan86,87, and NOD1�/� macrophages

failed to produce cytokines in response to iE-DAP87. In contrast, NOD2 is

a receptor for muramyl-dipeptide (MDP) derived from bacterial peptidogly-

can (PGN)88. A missense point mutation in the human Nod2 gene is

correlated with susceptibility to Crohn’s disease, an inflammatory bowel

disease. Overexpression of a mutant NOD2 in cells failed to induce NF-kB

activation89. In addition, NOD2�/� mice did not respond to MDP. However,

the mechanism for the development of this inflammatory disease is still

controversial. NOD2�/� mice were reported to show defective induction of

intestinal anti-microbial peptides after oral bacterial infection90, and were

susceptible to mucosal bacterial infection, thereby explaining the role

of NOD2 in the development of Crohn’s disease90. Another study reported

that NOD2 suppressed TLR2-mediated activation of NF-kB in response to

PGN91. NOD2�/� mice showed enhanced IL-12 production in response to

PGN stimulation, which may explain the mechanism for how NOD2

controls the susceptibility to this disease91. Furthermore, mice with a muta-

tion corresponding to that of Crohn’s disease showed enhanced NF-kB

activation in response to MDP, and increased susceptibility to dextran

sodium sulfate (DSS)-induced colitis92. The mutant mice exhibited elevated

NF-kB activation in response to MDP. Given the controversy between these

�37

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

separate studies, future studies are required to clarify the mechanism for the

function of NOD2 in intestinal immune cells. Ligand binding to NOD1 and

NOD2 causes their oligomerization and results in NF-kB activation through

the recruitment of RIP2/Rip-like interacting caspase-like apoptosis-regula-

tory protein kinase (RICK), a serine/threonine kinase93. NODs associate

with RIP2/RICK through their CARD domains by homophilic interactions.

It has been revealed that proteins with a nucleotide-binding oligomerization

domain (NOD) and a LRR domain form a large group, called the NOD-LRR

family94. The members of this family share a tripartite domain structure

consisting of C-terminal LRR motifs, a central NOD and N-terminal protein–

protein interaction motifs, such as CARDs, pyrin domains or a TIR

domain94. Although the number of NOD-LRR family members is continuing

to increase, the functions of members other than NOD1 and NOD2 have not

yet been clarified.

2.7 DISTURBANCE OF TLR SIGNALING BY PATHOGENS

Activation of the immune system is a threat to the survival of pathogens.

Therefore, viruses and bacteria have developed elaborate mechanisms to

escape from host immune surveillance. Pathogens modify and/or inhibit

host immune responses, such as the induction of IFNs and proinflammatory

cytokines, and inhibit apoptosis.

The TLR system is a target of viral immune disturbance. Vaccinia

virus encodes the TIR domain-containing proteins A46R and A52R95,96.

A46R targets host TIR domain-containing adaptors and suppresses TLR- or

IL-1R-induced NF-kB activation. Similarly, A52R suppresses TLR-mediated

signaling. However, A46R and A52R each have distinct roles in TLR3-

mediated signaling: A46R inhibits IRF-3, whereas A52R blocks NF-kB96.

Vaccinia virus also encodes another protein, N1L, that antagonizes TLR

signaling at the level of IkB kinases and TBK197.

Hepatitis C virus (HCV) also has a mechanism for suppressing TLR

signaling. HCV NS3/4A protease was shown to block IRF-3 activation by

cleaving TRIF98. This cleavage decreases the abundance of TRIF and inhibits

poly I:C-mediated activation of IFN responses. The RIG-I system is

considered to be another viral target for immune evasion, and NS3/4A was

also able to inhibit RIG-I mediated responses in a TRIF-independent

manner99. The V proteins of paramyxoviruses associate with Mda-5, and

inhibit dsRNA-induced activation of the IFNb promoter83.

�

38

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

Bacteria and fungi exploit the TLR system to evade host immune

responses. For instance, Mycobacterium tuberculosis avoids being killed by

macrophages by inhibiting IFN-g-mediated signaling. Prolonged signaling

with a 19-kDa lipoprotein from Mycobacterium, which stimulates TLR2,

inhibits IFNg production and major histocompatibility complex (MHC) class

II antigen processing activity100. These findings suggest that, at least in part,

TLR2 signaling promotes evasion of T cell responses and the persistence of

Mycobacterium. Candida albicans was also reported to exploit TLR2 signaling

to suppress immunity. TLR2�/� mice were more resistant to systemic

Candida infection and IL-10 release was impaired in TLR2�/� mice101.

These observations indicate that the TLR signaling pathways and

additional signaling pathways involved in pathogen recognition are inhibited

and exploited by pathogens for their survival.

2.8 PERSPECTIVES

In this chapter, we have focused on the signaling pathways activated in

response to exposure to pathogens. Although all TLRs possess a conserved

TIR domain that is critical for the activation of intracellular signaling, each

TLR induces a distinct response. This can partly be explained by the

recruitment of different TIR domain-containing adaptor molecules to each

receptor. In addition, cell type-specific recruitment of distinct signaling

machinery is also responsible for selective IFN-a production.

Recent extensive studies have identified ligands for most of the TLRs as

well as their signaling pathways. TLR2 and TLR5, which recognize bacterial,

but not viral, components, are completely dependent on MyD88 for

intracellular signaling. Therefore, the production of proinflammatory

cytokines, but not IFNs, is induced in response to TLR2 and TLR5 ligands.

In contrast, TLR3 and TLR7 recognize virus-specific components, while

TLR4 and TLR9 can respond to both bacteria and viruses. TLRs that

recognize viral components induce the production of type I IFNs in response

to proinflammatory cytokines. This adaptive response depends on the type

of pathogen and can be explained by the adaptor molecules recruited to each

TLR or by a cell type-specific mechanism.

Recent studies have rapidly identified the TLR signaling pathways

activated by distinct TLR ligands. In addition, intracellular receptors for

pathogens and their signaling pathways are beginning to be identified.

However, the complete mechanisms involved in immune-recognition and

signaling are far from comprehensively understood. In addition, immune

�39

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

cells utilize different signaling pathways in a cell type-specific manner.

Future studies are necessary to clarify the mechanisms and develop

techniques for manipulating the pathways to improve therapeutic treat-

ments for infectious diseases.

REFERENCES

1. Akira, S. and K. Takeda (2004). Toll-like receptor signalling. Nat. Rev.

Immunol. 4: 499–511.

2. Beutler, B. (2004). Inferences, questions and possibilities in Toll-like

receptor signalling. Nature 430: 257–63.

3. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. Van Huffel, X. Du,

D. Birdwell, E. Alejos, M. Silva, C. Galanos, M. Freudenberg,

P. Ricciardi-Castagnoli, B. Layton, and B. Beutler (1998). Defective LPS

signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene.

Science 282: 2085–8.

4. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda,

K. Takeda, and S. Akira (1999). Cutting edge: Toll-like receptor 4 (TLR4)-

deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4

as the Lps gene product. J. Immunol. 162: 3749–52.

5. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa,

K. Takeda, and S. Akira (1999). Differential roles of TLR2 and TLR4 in

recognition of Gram-negative and Gram-positive bacterial cell wall

components. Immunity 11: 443–51.

6. Takeuchi, O., T. Kawai, P. F. Muhlradt, M. Morr, J. D. Radolf, A. Zychlinsky,

K. Takeda, and S. Akira (2001). Discrimination of bacterial lipoproteins by

Toll-like receptor 6. Int. Immunol. 13: 933–40.

7. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong,

R. L. Modlin, and S. Akira (2002). Cutting edge: role of Toll-like receptor 1 in

mediating immune response to microbial lipoproteins. J. Immunol.

169: 10–14.

8. Alexopoulou, L., V. Thomas, M. Schnare, Y. Lobet, J. Anguita, R. T. Schoen,

R. Medzhitov, E. Fikrig, and R. A. Flavell (2002). Hyporesponsiveness to

vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and

TLR2-deficient mice. Nat. Med. 8: 878–84.

9. Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C. Yi, D. R. Goodlett,

J. K. Eng, S. Akira, D. M. Underhill, and A. Aderem (2001). The innate

immune response to bacterial flagellin is mediated by Toll-like receptor 5.

Nature 410: 1099–103.

�

40

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

10. Zhang, D., G. Zhang, M. S. Hayden, M. B. Greenblatt, C. Bussey,

R. A. Flavell, and S. Ghosh (2004). A toll-like receptor that prevents

infection by uropathogenic bacteria. Science 303: 1522–6.

11. Yarovinsky, F., D. Zhang, J. F. Andersen, G. L. Bannenberg, C. N. Serhan,

M. S. Hayden, S. Hieny, F. S. Sutterwala, R. A. Flavell, S. Ghosh, and

A. Sher (2005). TLR11 Activation of dendritic cells by a protozoan

profilin-like protein. Science 308: 1626–9.

12. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell (2001).

Recognition of double-stranded RNA and activation of NF-kappaB by

Toll-like receptor 3. Nature 413: 732–8.

13. Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning,

S. Akira, G. Lipford, H. Wagner, and S. Bauer (2004). Species-specific

recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science

303: 1526–9.

14. Diebold, S. S., T. Kaisho, H. Hemmi, S. Akira, and C. Reis e Sousa (2004).

Innate antiviral responses by means of TLR7-mediated recognition of

single-stranded RNA. Science 303: 1529–31.

15. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo,

M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, and S. Akira

(2000). A Toll-like receptor recognizes bacterial DNA. Nature 408:

740–5.

16. Lund, J., A. Sato, S. Akira, R. Medzhitov, and A. Iwasaki (2003). Toll-like

receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid

dendritic cells. J. Exp. Med. 198: 513–20.

17. Krug, A., G. D. Luker, W. Barchet, D. A. Leib, S. Akira, and

M. Colonna (2004). Herpes simplex virus type 1 activates murine

natural interferon-producing cells through Toll-like receptor 9. Blood

103: 1433–7.

18. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks,

C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, and D. T. Golenbock (2004).

TLR9 signals after translocating from the ER to CpG DNA in the lysosome.

Nat. Immunol. 5: 190–8.

19. Couillault, C., N. Pujol, J. Reboul, L. Sabatier, J. F. Guichou, Y. Kohara,

and J. J. Ewbank (2004). TLR-independent control of innate immunity in

Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an

ortholog of human SARM. Nat. Immunol. 5: 488–94.

20. Liberati, N. T., K. A. Fitzgerald, D. H. Kim, R. Feinbaum, D. T. Golenbock,

and F. M. Ausubel (2004). Requirement for a conserved Toll/interleukin-1

resistance domain protein in the Caenorhabditis elegans immune response.

Proc. Natl Acad. Sci. U S A 101: 6593–8.

�41

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

21. Muzio, M., J. Ni, P. Feng, and V. M. Dixit (1997). IRAK (Pelle) family

member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling.

Science 278: 1612–15.

22. Wesche, H., W. J. Henzel, W. Shillinglaw, S. Li, and Z. Cao (1997). MyD88:

an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7:

837–47.

23. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami,

K. Nakanishi, and S. Akira (1998). Targeted disruption of the MyD88

gene results in loss of IL-1- and IL-18-mediated function. Immunity

9: 143–50.

24. Kawai, T., O. Adachi, T. Ogawa, K. Takeda, and S. Akira (1999).

Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:

115–22.

25. Takeuchi, O., K. Takeda, K. Hoshino, O. Adachi, T. Ogawa, and S. Akira

(2000). Cellular responses to bacterial cell wall components are

mediated through MyD88-dependent signaling cascades. Int. Immunol.

12: 113–17.

26. Horng, T., G. M. Barton, and R. Medzhitov (2001). TIRAP: an adapter

molecule in the Toll signaling pathway. Nat. Immunol. 2: 835–41.

27. Fitzgerald, K. A., E. M. Palsson-McDermott, A. G. Bowie, C. A. Jefferies,

A. S. Mansell, G. Brady, E. Brint, A. Dunne, P. Gray, M. T. Harte,

D. McMurray, D. E. Smith, J. E. Sims, T. A. Bird, and L. A. O’Neill (2001).

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal

transduction. Nature 413: 78–83.

28. Horng, T., G. M. Barton, R. A. Flavell, and R. Medzhitov (2002). The adaptor

molecule TIRAP provides signalling specificity for Toll-like receptors.

Nature 420: 329–33.

29. Yamamoto, M., S. Sato, H. Hemmi, H. Sanjo, S. Uematsu, T. Kaisho,

K. Hoshino, O. Takeuchi, M. Kobayashi, T. Fujita, K. Takeda, and S. Akira

(2002). Essential role for TIRAP in activation of the signalling cascade

shared by TLR2 and TLR4. Nature 420: 324–9.

30. Li, S., A. Strelow, E. J. Fontana, and H. Wesche (2002). IRAK-4: a novel

member of the IRAK family with the properties of an IRAK-kinase.

Proc. Natl Acad. Sci. U S A 99: 5567–72.

31. Suzuki, N., S. Suzuki, G. S. Duncan, D. G. Millar, T. Wada, C. Mirtsos,

H. Takada, A. Wakeham, A. Itie, S. Li, J. M. Penninger, H. Wesche,

P. S. Ohashi, T. W. Mak, and W. C. Yeh (2002). Severe impairment

of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4.

Nature 416: 750–6.

�

42

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

32. Thomas, J. A., J. L. Allen, M. Tsen, T. Dubnicoff, J. Danao, X. C. Liao,

Z. Cao, and S. A. Wasserman (1999). Impaired cytokine signaling in mice

lacking the IL-1 receptor-associated kinase. J. Immunol. 163: 978–84.

33. Swantek, J. L., M. F. Tsen, M. H. Cobb, and J. A. Thomas (2000).

IL-1 receptor-associated kinase modulates host responsiveness to

endotoxin. J. Immunol. 164: 4301–6.

34. Jiang, Z., J. Ninomiya-Tsuji, Y. Qian, K. Matsumoto, and X. Li (2002).

Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced

signaling complexes phosphorylate TAK1 and TAB2 at the plasma

membrane and activate TAK1 in the cytosol. Mol. Cell Biol. 22: 7158–67.

35. Deng, L., C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter,

C. Pickart, and Z. J. Chen (2000). Activation of the IkappaB kinase complex

by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and

a unique polyubiquitin chain. Cell 103: 351–61.

36. Wang, C., L. Deng, M. Hong, G. R. Akkaraju, J. Inoue, and Z. J. Chen

(2001). TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature

412: 346–51.

37. Ishitani, T., G. Takaesu, J. Ninomiya-Tsuji, H. Shibuya, R. B. Gaynor, and

K. Matsumoto (2003). Role of the TAB2-related protein TAB3 in IL-1

and TNF signaling. EMBO J. 22: 6277–88.

38. Kanayama, A., R. B. Seth, L. Sun, C. K. Ea, M. Hong, A. Shaito, Y. H. Chiu,

L. Deng, and Z. J. Chen (2004). TAB2 and TAB3 activate the NF-kappaB

pathway through binding to polyubiquitin chains. Mol. Cell 15: 535–48.

39. Sanjo, H., K. Takeda, T. Tsujimura, J. Ninomiya-Tsuji, K. Matsumoto, and

S. Akira (2003). TAB2 is essential for prevention of apoptosis in fetal liver

but not for interleukin-1 signaling. Mol. Cell Biol. 23: 1231–8.

40. Hayden, M. S. and S. Ghosh (2004). Signaling to NF-kappaB. Genes Dev.

18: 2195–224.

41. McDermott, E. P. and L. A. O’Neill (2002). Ras participates in the activation

of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6,

and TAK-1. J. Biol. Chem. 277: 7808–15.

42. Matsuzawa, A., K. Saegusa, T. Noguchi, C. Sadamitsu, H. Nishitoh,

S. Nagai, S. Koyasu, K. Matsumoto, K. Takeda, and H. Ichijo (2005).

ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively

required for TLR4-mediated innate immunity. Nat. Immunol. 6: 587–92.

43. Takaoka, A., H. Yanai, S. Kondo, G. Duncan, H. Negishi, T. Mizutani,

S. Kano, K. Honda, Y. Ohba, T. W. Mak, and T. Taniguchi (2005).

Integral role of IRF-5 in the gene induction programme activated by

Toll-like receptors. Nature 434: 243–9.

�43

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

44. Schoenemeyer, A., B. J. Barnes, M. E. Mancl, E. Latz, N. Goutagny,

P. M. Pitha, K. A. Fitzgerald, and D. T. Golenbock (2005). The interferon

regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling.

J. Biol. Chem. 280: 17005–12.

45. Brint, E. K., D. Xu, H. Liu, A. Dunne, A. N. McKenzie, L. A. O’Neill, and

F. Y. Liew (2004). ST2 is an inhibitor of interleukin 1 receptor and Toll-like

receptor 4 signaling and maintains endotoxin tolerance. Nat. Immunol.

5: 373–9.

46. Divanovic, S., A. Trompette, S. F. Atabani, R. Madan, D. T. Golenbock,

A. Visintin, R. W. Finberg, A. Tarakhovsky, S. N. Vogel, Y. Belkaid,

E. A. Kurt-Jones, and C. L. Karp (2005). Negative regulation of Toll-like

receptor 4 signaling by the Toll-like receptor homolog RP105. Nat.

Immunol. 6: 571–8.

47. Nagai, Y., T. Kobayashi, Y. Motoi, K. Ishiguro, S. Akashi, S. Saitoh,

Y. Kusumoto, T. Kaisho, S. Akira, M. Matsumoto, K. Takatsu, and K.

Miyake (2005). The radioprotective 105/MD-1 complex links TLR2 and

TLR4/MD-2 in antibody response to microbial membranes. J. Immunol.

174: 7043–9.

48. Chuang, T. H. and R. J. Ulevitch (2004). Triad3A, an E3 ubiquitin-protein

ligase regulating Toll-like receptors. Nat. Immunol. 5: 495–502.

49. Hamerman, J. A., N. K. Tchao, C. A. Lowell, and L. L. Lanier (2005).

Enhanced Toll-like receptor responses in the absence of signaling adaptor

DAP12. Nat. Immunol. 6: 579–86.

50. Burns, K., S. Janssens, B. Brissoni, N. Olivos, R. Beyaert, and J. Tschopp

(2003). Inhibition of interleukin 1 receptor/Toll-like receptor signaling

through the alternatively spliced, short form of MyD88 is due to its failure

to recruit IRAK-4. J. Exp. Med. 197: 263–8.

51. Burns, K., J. Clatworthy, L. Martin, F. Martinon, C. Plumpton, B. Maschera,

A. Lewis, K. Ray, J. Tschopp, and F. Volpe (2000). Tollip, a new component

of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat. Cell Biol.

2: 346–51.

52. Zhang, G. and S. Ghosh (2002). Negative regulation of Toll-like

receptor-mediated signaling by Tollip. J. Biol. Chem. 277: 7059–65.

53. Boone, D. L., E. E. Turer, E. G. Lee, R. C. Ahmad, M. T. Wheeler,

C. Tsui, P. Hurley, M. Chien, S. Chai, O. Hitotsumatsu, E. McNally,

C. Pickart, and A. Ma (2004). The ubiquitin-modifying enzyme A20 is

required for termination of Toll-like receptor responses. Nat. Immunol.

5: 1052–60.

�

44

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

54. Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda, and

S. Akira (2002). Cutting edge: a novel Toll/IL-1 receptor domain-containing

adapter that preferentially activates the IFN-beta promoter in the Toll-like

receptor signaling. J. Immunol. 169: 6668–72.

55. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, and T. Seya (2003).

TICAM-1, an adaptor molecule that participates in Toll-like receptor

3-mediated interferon-beta induction. Nat. Immunol. 4: 161–7.

56. Sharma, S., B. R. tenOever, N. Grandvaux, G. P. Zhou, R. Lin, and J. Hiscott

(2003). Triggering the interferon antiviral response through an IKK-related

pathway. Science 300: 1148–51.

57. Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz,

D. T. Golenbock, A. J. Coyle, S. M. Liao, and T. Maniatis (2003).

IKKepsilon and TBK1 are essential components of the IRF3 signaling

pathway. Nat. Immunol. 4: 491–6.

58. Hemmi, H., O. Takeuchi, S. Sato, M. Yamamoto, T. Kaisho, H. Sanjo,

T. Kawai, K. Hoshino, K. Takeda, and S. Akira (2004). The roles of two

IkappaB kinase-related kinases in lipopolysaccharide and double stranded

RNA signaling and viral infection. J. Exp. Med. 199: 1641–50.

59. McWhirter, S. M., K. A. Fitzgerald, J. Rosains, D. C. Rowe, D. T. Golenbock,

and T. Maniatis (2004). IFN-regulatory factor 3-dependent gene expression

is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc. Natl Acad.

Sci. U S A 101: 233–8.

60. Perry, A. K., E. K. Chow, J. B. Goodnough, W. C. Yeh, and G. Cheng (2004).

Differential requirement for TANK-binding kinase-1 in type I interferon

responses to Toll-like receptor activation and viral infection. J. Exp. Med.

199: 1651–8.

61. Yoneyama, M., W. Suhara, Y. Fukuhara, M. Fukuda, E. Nishida, and

T. Fujita (1998). Direct triggering of the type I interferon system by virus

infection: activation of a transcription factor complex containing IRF-3 and

CBP/p300. EMBO J. 17: 1087–95.

62. Sakaguchi, S., H. Negishi, M. Asagiri, C. Nakajima, T. Mizutani,

A. Takaoka, K. Honda, and T. Taniguchi (2003). Essential role of IRF-3 in

lipopolysaccharide-induced interferon-beta gene expression and endotoxin

shock. Biochem. Biophys. Res. Commun. 306: 860–6.

63. Honda, K., H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani,

N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi (2005).

IRF-7 is the master regulator of type-I interferon-dependent immune

responses. Nature 434: 772–7.

�45

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

64. Sato, S., M. Sugiyama, M. Yamamoto, Y. Watanabe, T. Kawai, K. Takeda,

and S. Akira (2003). Toll/IL-1 receptor domain-containing adaptor

inducing IFN-beta (TRIF) associates with TNF receptor-associated factor 6

and TANK-binding kinase 1, and activates two distinct transcription factors,

NF-kappa B and IFN-regulatory factor-3, in the Toll-like receptor signaling.

J. Immunol. 171: 4304–10.

65. Meylan, E., K. Burns, K. Hofmann, V. Blancheteau, F. Martinon,

M. Kelliher, and J. Tschopp (2004). RIP1 is an essential mediator of

Toll-like receptor 3-induced NF-kappaB activation. Nat. Immunol.

5: 503–7.

66. Han, K. J., X. Su, L. G. Xu, L. H. Bin, J. Zhang, and H. B. Shu (2004).

Mechanisms of the TRIF-induced interferon-stimulated response element

and NF-kappaB activation and apoptosis pathways. J. Biol. Chem.

279: 15652–61.

67. Kaiser, W. J. and M. K. Offermann (2005). Apoptosis induced by the

toll-like receptor adaptor TRIF is dependent on its receptor interacting

protein homotypic interaction motif. J. Immunol. 174: 4942–52.

68. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo,

O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, and S. Akira (2003). Role

of adaptor TRIF in the MyD88-independent toll-like receptor signaling

pathway. Science 301: 640–3.

69. Hirotani, T., M. Yamamoto, Y. Kumagai, S. Uematsu, I. Kawase,

O. Takeuchi, and S. Akira (2005). Regulation of lipopolysaccharide-

inducible genes by MyD88 and Toll/IL-1 domain containing adaptor

inducing IFN-beta. Biochem. Biophys. Res. Commun. 328: 383–92.

70. Fitzgerald, K. A., D. C. Rowe, B. J. Barnes, D. R. Caffrey, A. Visintin, E. Latz,

B. Monks, P. M. Pitha, and D. T. Golenbock (2003). LPS-TLR4 signaling

to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF.

J. Exp. Med. 198: 1043–55.

71. Yamamoto, M., S. Sato, H. Hemmi, S. Uematsu, K. Hoshino, T. Kaisho,

O. Takeuchi, K. Takeda, and S. Akira (2003). TRAM is specifically involved

in the Toll-like receptor 4-mediated MyD88-independent signaling

pathway. Nat. Immunol. 4: 1144–50.

72. Liu, Y. J. (2005). IPC: Professional type 1 interferon-producing cells and

plasmacytoid dendritic cell precursors. Annu. Rev. Immunol. 23: 275–306.

73. Hemmi, H., T. Kaisho, K. Takeda, and S. Akira (2003). The roles of

Toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic

subunit in the effects of two distinct CpG DNAs on dendritic cell subsets.

J. Immunol. 170: 3059–64.

�

46

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

74. Hochrein, H., B. Schlatter, M. O’Keeffe, C. Wagner, F. Schmitz,

M. Schiemann, S. Bauer, M. Suter, and H. Wagner (2004). Herpes

simplex virus type-1 induces IFN-alpha production via Toll-like receptor

9-dependent and -independent pathways. Proc. Natl Acad. Sci. U S A

101: 11416–21.

75. Kawai, T., S. Sato, K. J. Ishii, C. Coban, H. Hemmi, M. Yamamoto, K. Terai,

M. Matsuda, J. Inoue, S. Uematsu, O. Takeuchi, and S. Akira (2004).

Interferon-alpha induction through Toll-like receptors involves a direct

interaction of IRF7 with MyD88 and TRAF6. Nat. Immunol. 5: 1061–8.

76. Honda, K., H. Yanai, T. Mizutani, H. Negishi, N. Shimada, N. Suzuki,

Y. Ohba, A. Takaoka, W. C. Yeh, and T. Taniguchi (2004). Role of

a transductional-transcriptional processor complex involving MyD88

and IRF-7 in Toll-like receptor signaling. Proc. Natl Acad. Sci. U SA

101: 15416–21.

77. Uematsu, S., S. Sato, M. Yamamoto, T. Hirotani, H. Kato, F. Takeshita,

M. Matsuda, C. Coban, K. J. Ishii, T. Kawai, O. Takeuchi, and S. Akira

(2005). Interleukin-1 receptor-associated kinase-1 plays an essential role

for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha}

induction. J. Exp. Med. 201: 915–23.

78. Kerkmann, M., S. Rothenfusser, V. Hornung, A. Towarowski, M. Wagner,

A. Sarris, T. Giese, S. Endres, and G. Hartmann (2003). Activation with

CpG-A and CpG-B oligonucleotides reveals two distinct regulatory

pathways of type I IFN synthesis in human plasmacytoid dendritic cells.

J. Immunol. 170: 4465–74.

79. Honda, K., Y. Ohba, H. Yanai, H. Negishi, T. Mizutani, A. Takaoka, C. Taya,

and T. Taniguchi (2005). Spatiotemporal regulation of MyD88-IRF-7

signalling for robust type-I interferon induction. Nature 434: 1035–40.

80. Diebold, S. S., M. Montoya, H. Unger, L. Alexopoulou, P. Roy, L. E. Haswell,

A. Al-Shamkhani, R. Flavell, P. Borrow, and C. Reis e Sousa (2003). Viral

infection switches non-plasmacytoid dendritic cells into high interferon

producers. Nature 424: 324–8.

81. Smith, E. J., I. Marie, A. Prakash, A. Garcia-Sastre, and D. E. Levy (2001).

IRF3 and IRF7 phosphorylation in virus-infected cells does not require

double-stranded RNA-dependent protein kinase R or Ikappa B kinase

but is blocked by Vaccinia virus E3L protein. J. Biol. Chem. 276: 8951–7.

82. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu,

T. Imaizumi, M. Miyagishi, K. Taira, S. Akira, and T. Fujita (2004).

The RNA helicase RIG-I has an essential function in double-stranded

RNA-induced innate antiviral responses. Nat. Immunol. 5: 730–7.

�47

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

83. Andrejeva, J., K. S. Childs, D. F. Young, T. S. Carlos, N. Stock,

S. Goodbourn, and R. E. Randall (2004). The V proteins of

paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and

inhibit its activation of the IFN-beta promoter. Proc. Natl Acad. Sci.

U SA 101: 17264–9.

84. Kato, H., S. Sato, M. Yoneyama, M. Yamamoto, S. Uematsu, K. Matsui,

T. Tsujimura, K. Takada, T. Fujita, O. Takeuchi, and S. Akira (2005). Cell

type-specific involvement of RIG-I in antiviral response. Immunity

23: 19–28.

85. Balachandran, S., E. Thomas, and G. N. Barber (2004). A FADD-

dependent innate immune mechanism in mammalian cells. Nature

432: 401–5.

86. Girardin, S. E., I. G. Boneca, L. A. Carneiro, A. Antignac, M. Jehanno,

J. Viala, K. Tedin, M. K. Taha, A. Labigne, U. Zahringer, A. J. Coyle,

P. S. DiStefano, J. Bertin, P. J. Sansonetti, and D. J. Philpott (2003). Nod1

detects a unique muropeptide from Gram-negative bacterial peptidoglycan.

Science 300: 1584–7.

87. Chamaillard, M., M. Hashimoto, Y. Horie, J. Masumoto, S. Qiu, L. Saab,

Y. Ogura, A. Kawasaki, K. Fukase, S. Kusumoto, M. A. Valvano, S. J. Foster,

T. W. Mak, G. Nunez, and N. Inohara (2003). An essential role for NOD1

in host recognition of bacterial peptidoglycan containing diaminopimelic

acid. Nat. Immunol. 4: 702–7.

88. Girardin, S. E., I. G. Boneca, J. Viala, M. Chamaillard, A. Labigne,

G. Thomas, D. J. Philpott, and P. J. Sansonetti (2003). Nod2 is a general

sensor of peptidoglycan through muramyl dipeptide (MDP) detection.

J. Biol. Chem. 278: 8869–72.

89. Ogura, Y., D. K. Bonen, N. Inohara, D. L. Nicolae, F. F. Chen, R. Ramos,

H. Britton, T. Moran, R. Karaliuskas, R. H. Duerr, J. P. Achkar, S. R. Brant,

T. M. Bayless, B. S. Kirschner, S. B. Hanauer, G. Nunez, and J. H. Cho

(2001). A frameshift mutation in NOD2 associated with susceptibility to

Crohn’s disease. Nature 411: 603–6.

90. Kobayashi, K. S., M. Chamaillard, Y. Ogura, O. Henegariu, N. Inohara,

G. Nunez, and R. A. Flavell (2005). Nod2-dependent regulation of

innate and adaptive immunity in the intestinal tract. Science

307: 731–4.

91. Watanabe, T., A. Kitani, P. J. Murray, and W. Strober (2004). NOD2 is

a negative regulator of Toll-like receptor 2-mediated T helper type 1

responses. Nat. Immunol. 5: 800–8.

�

48

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

92. Maeda, S., L. C. Hsu, H. Liu, L. A. Bankston, M. Iimura, M. F. Kagnoff,

L. Eckmann, and M. Karin (2005). Nod2 mutation in Crohn’s

disease potentiates NF-kappaB activity and IL-1beta processing.

Science 307: 734–8.

93. Kobayashi, K., N. Inohara, L. D. Hernandez, J. E. Galan, G. Nunez,

C. A. Janeway, R. Medzhitov, and R. A. Flavell (2002). RICK/Rip2/

CARDIAK mediates signalling for receptors of the innate and adaptive

immune systems. Nature 416: 194–9.

94. Inohara, N., M. Chamaillard, C. McDonald, and G. Nunez (2005).

NOD-LRR proteins: role in host-microbial interactions and inflammatory

disease. Annu. Rev. Biochem. 74: 355–83.

95. Bowie, A., E. Kiss-Toth, J. A. Symons, G. L. Smith, S. K. Dower, and

L. A. O’Neill (2000). A46R and A52R from vaccinia virus are antagonists of

host IL-1 and toll-like receptor signaling. Proc. Natl Acad. Sci. U S A

97: 10162–7.

96. Stack, J., I. R. Haga, M. Schroder, N. W. Bartlett, G. Maloney, P. C. Reading,

K. A. Fitzgerald, G. L. Smith, and A. G. Bowie (2005). Vaccinia virus protein

A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contri-

butes to virulence. J. Exp. Med. 201: 1007–18.

97. DiPerna, G., J. Stack, A. G. Bowie, A. Boyd, G. Kotwal, Z. Zhang, S. Arvikar,

E. Latz, K. A. Fitzgerald, and W. L. Marshall (2004). Poxvirus protein

N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB

by the tumor necrosis factor superfamily of receptors, and inhibits

NF-kappaB and IRF3 signaling by toll-like receptors. J. Biol. Chem.

279: 36570–8.

98. Li, K., E. Foy, J. C. Ferreon, M. Nakamura, A. C. Ferreon, M. Ikeda, S. C. Ray,

M. Gale, Jr., and S. M. Lemon (2005). Immune evasion by hepatitis C virus

NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor

protein TRIF. Proc. Natl Acad. Sci. U S A 102: 2992–7.

99. Breiman, A., N. Grandvaux, R. Lin, C. Ottone, S. Akira, M. Yoneyama, T.

Fujita, J. Hiscott, and E. F. Meurs (2005). Inhibition of RIG-I-dependent

signaling to the interferon pathway during hepatitis C virus expression and

restoration of signaling by IKKepsilon. J. Virol. 79: 3969–78.

100. Fortune, S. M., A. Solache, A. Jaeger, P. J. Hill, J. T. Belisle, B. R. Bloom,

E. J. Rubin, and J. D. Ernst (2004). Mycobacterium tuberculosis inhibits

macrophage responses to IFN-gamma through myeloid differentiation

factor 88-dependent and -independent mechanisms. J. Immunol.

172: 6272–80.

�49

TO

LL-L

IKE

RE

CE

PT

OR

SIG

NA

LIN

G

101. Netea, M. G., R. Sutmuller, C. Hermann, C. A. Van der Graaf,

J. W. Van der Meer, J. H. van Krieken, T. Hartung, G. Adema, and

B. J. Kullberg (2004). Toll-like receptor 2 suppresses immunity against

Candida albicans through induction of IL-10 and regulatory T cells.

J. Immunol. 172: 3712–18.

�

50

O.

TA

KE

UC

HI

AN

DS.

AK

IRA

CHAPTER 3

MHC class I and II pathways for presentationand cross-presentation of bacterial antigens

Laurence Bougneres-Vermont and Pierre GuermonprezInstitut Curie

3.1 DENDRITIC CELLS PRIME ANTI-BACTERIAL CD4þ AND CD8þ

T CELLS IN VIVO

It is now widely accepted that dendritic cells (DCs) are crucially required

for the priming of T cell responses1,2. Major histo-compatibility complex

(MHC) class I and class II presentation pathways ensure the priming

of CD8þ and CD4þ T cell, respectively. They thus represent major

checkpoints for the induction of adaptative protective immunity toward

intracellular bacteria. In this chapter, we will focus on the basic cell

biology and physiological regulation of these pathways in the context of

bacterial infection. In accordance with the literature, we will refer to ‘‘cross

presentation’’ for MHC class I pathways involved in the presentation of

non cytosolic antigens.

Listeria3,4, Mycobacteria5–7 and Salmonella8–12 were shown to actually

infect DCs in situ. Some studies have addressed the capacity of DCs

purified from infected animals to activate in vitro T cells specific for

bacteria-encoded antigens. Intravenous infection of mice with Salmonella10

and with Mycobacterium bovis BCG (bacillus Calmette-Guerin)7 leads to

the infection of both spleen DC subsets (CD8a� CD11cþ and CD8aþ

CD11cþ). Both subsets display some MHC class I and II complexes

formed after the processing of bacteria-encoded antigens7,10. Bacterial

infection may also promote apoptosis, resulting in the delivery of bacterial

antigens to DCs upon the phagocytosis of infected apoptotic bodies (see

Section 3.5)13.

Whatever may be the mechanism (infection or dead cells cross

presentation), the absolute requirement of DCs to induce anti-bacterial

T cell priming was elegantly demonstrated in the Listeria model.

Using bone-marrow chimera, Lenz et al. first demonstrated that antigen

presenting cells required for the priming of Listeria-specific CD8þ T cells

were from hematopoietic origin14. Jung et al. developed a transgenic mouse

expressing selectively the diphtheria toxin receptor at the surface of DCs

(CD11c-DTR)15. DC-depleted animals upon diphtheria toxin injection were

no longer capable of mounting a CD8þ T cell response against Listeria. This

study established unambiguously, at least for Listeria, the crucial role of DCs

in anti-bacterial T cell priming in vivo.

3.2 MHC CLASS I PATHWAYS

3.2.1 The cytosolic pathway for MHC class I presentation

Basic mechanisms of the cytosolic pathway (Figure 3.1)

MHC class I molecules bind and present peptides around ten amino acids

to CD8þ T lymphocytes. These peptides are generally derived from cytosolic

proteins and generated upon the coordination of various cytosolic and

noncytosolic protease activity. Defective ribosomal products (DRIPs) may

provide the major source of antigens for the cytosolic pathway16.

Ubiquitinylation of proteins or DRIPs target them to the proteasome

for proteolytic degradation. The proteasome complex play a major role in

the cytosolic proteolytic process17. Proteasomes are generally required for

generation of the C-terminus of the peptides that are found loaded

on MHC class I molecules. Under transcriptional activation (by IFNs,

for example), three proteasomal subunits are replaced by their ‘‘immuno-

logical’’ homologues LMP2, LMP7 (encoded in the MHC) and MECL1

thus forming immunoproteasome complexes. Immunoproteasome forma-

tion may further increase the generation of ‘‘presentable’’ peptides by

promoting the cleavage after basic or hydrophobic residues. TPPII and

other cytosolic aminopeptidases participate in some cases to peptide

trimming18. Then, peptides derived from cytosolic proteolysis are trans-

located into endoplasmic reticulum (ER) lumen by transporters associated

with antigen processing (TAP). In the ER lumen they can undergo

further N-terminal trimming by aminopeptidases such as endoplasmic

reticulum aminopeptidase (ERAP)1 or ERAP217,18. Optimal length peptides

are then associated to MHC class I molecules by the MHC class I complex

loading19. The MHC-encoded tapasin chaperone acts as a molecular

bridge between the neosynthesized MHC class I and TAP transporters.

Chaperones, like calnexin and calreticulin, and oxydoreductases like Erp57,

cooperate for the assembly of the trimeric complexes containing MHC

�

52

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

class I heavy chain, b2-microglobulin and antigenic peptides. Consequently,

MHC class I-peptides traffic along the secretory pathway to cell surface.

Although submitted to transcriptional regulation (notably by IFNs), this

pathway is operative in most cell types allowing the detection of intra-

cellular pathogens such as viruses and bacteria.

DCs specific features of the cytosolic pathway

Unlike many other cell types, immature DCs express significant levels

of immunoproteasomes20. Upon LPS-triggered maturation, Pierre’s labora-

tory characterized the transient appearance of agreggosome-like structures,

the DALIS (dendritic cells aggresome-like induced structures), in DCs

induced to mature by LPS21,22. DRIPs are targeted toward these organized

and dynamic structures that seem to exclude long-lived proteins21. Based

on this observation, the authors proposed that this process may reflect

a transient shutdown of the bulk production of endogenous peptides by

DRIP processing, thus favoring the loading of cross-presented peptides.

The transient shutdown of DRIP processing may also favor proteasomal

processing of bacterial proteins secreted in the cytosol of infected cells.

In support of this, DALIS are also induced in bacteria-faced macrophages23.

Later stages of DC maturation are characterized by the upregulation of

the MHC class I presentation machinery including immunoproteasomes,

TAP, b2 microglobulin and MHC class I heavy chain24. At the level of MHC

class I trafficking, Ackerman et al. showed that immature DCs transiently

retain MHC class I in Golgi-associated pools25, whereas the induction

of DC maturation accelerates their delivery to the cell surface. Unlike

MHC class II, MHC class I half life is modestly increased during DCs

maturation25–28. Production of MHC class I-peptide complexes is sustained

in mature DCs26.

Bacterial antigens secreted by live, cytosol-invading bacteria enter the

cytosolic pathway

Intracellular bacteria such as Listeria and Shigella enter the cytosol of

invading cells where they replicate. The Listeria listeriolysin LLO triggers

Listeria-containing vacuole lysis which results in escape of the whole

bacterium into the cytosolic compartment29. CD8þ T cells are major com-

ponents of the adaptative immune response against Listeria. Pioneering

cellular studies from Pamer’s lab analyzed the pathways leading to the

presentation of bacteria-derived peptides by MHC class I molecules29,30.

Using a biochemical approach, they identified and quantified three

�53

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

Fig

ure

3.1

.M

HC

Ian

dM

HC

IIp

rese

nta

tio

np

ath

way

sfo

rb

acte

rial

anti

gen

s.T

he

‘‘cla

ssic

al’’

cyto

soli

cM

HC

-Ip

ath

way

isin

volv

edin

pre

sen

tati

on

of

bac

teri

al

secr

eted

anti

gen

sd

eliv

ered

by

live

cyto

soli

cb

acte

ria

(Sec

tio

n3

.2.1

)an

dcr

oss

-pre

sen

tati

on

of

vacu

ola

rb

acte

ria-

der

ived

anti

gen

s(S

ecti

on3.2

.3).

Th

eE

R-

ph

ago

som

eT

AP

-dep

end

ent

pat

hw

ayfo

rM

HC

-Icr

oss

-pre

sen

tati

on

may

be

use

din

the

case

of

ER

-co

nta

inin

gb

acte

rial

vacu

ole

s(S

ecti

on3

.2.4

).T

he

TA

P-

ind

epen

dan

tp

hag

oly

soso

mal

pat

hw

ayis

use

dfo

rM

HC

-I-r

estr

icte

dcr

oss

-pre

sen

tati

on

of

som

eva

cuo

lar

bac

teri

a-d

eriv

edan

tig

ens

(Sec

tio

n3.2

.2).

Th

e

ph

ago

lyso

som

alp

ath

way

isin

volv

edin

MH

C-I

Ip

rese

nta

tio

no

fva

cuo

lar

bac

teri

a-d

eriv

edan

tig

ens

(Sec

tio

n3

.3.1

)an

dm

ayb

eu

sed

inth

eca

seo

fcy

toso

lic

bac

teri

ase

qu

este

red

by

the

auto

ph

agic

pat

hw

ay(S

ecti

on3

.3.2

).

major epitopes presented by H-2Kd MHC class I molecules at the surface

of infected cells. Two of them belong to the p60 secreted protein,

the third one belongs to the LLO. As shown by pharmacological inhibition,

the generation of these epitopes depends on proteasomal degradation of

Listeria neosynthesized proteins. Villanueva et al. quantified the efficiency

of p60 presentation: depending on the epitope, 3 or 30% of the p60 gave

rise to peptide-MHC complexes31,32. These yields are much higher than

those found for a substrate targeted to complete degradation: in this case 1

out of 2000 proteins gave rise to a peptide–MHC complex33. This difference

could rely on methodological discrepancies between the two studies.

Alternatively, the cytosolic innate response triggered by bacteria may

control the efficiency of antigen processing pathways.

Moreover, genetic insertion of N-terminus residues that target the

ubiquitin proteasome pathway decreased p60 protein half-life within the

cytosol, and consequently increased the presentation of epitopes derived

from it34. This elegant study demonstrated that bacterial protein pro-

teasomal degradation by the host-cell proteasome follow the same rules as

host cell proteins.

Do nonsecreted antigens enter the cytosolic pathway?

In a major study, Shen et al. investigated the role of Listeria antigen location

to T cell immunity35. Both secreted and nonsecreted antigens were shown

to suitably activate T cells in vivo suggesting that both were efficiently

presented by DCs in some way. Strikingly, only CD8þ Tcell responses against

secreted antigens were protective, suggesting that most of somatic infected

cells were not capable of processing nonsecreted antigens. Several hypo-

theses may explain why DCs can prime CD8þ T cells against nonsecreted

antigens. First, DCs may have a selective ability to kill and degrade a part

of cytosolic Listeria. This process may be linked to the autophagic pathway

that has recently been identified as a defense pathway against cytosolic

bacteria36–38. Second, a subset of DCs may limit the access of Listeria to

the cytosolic compartment and deliver Listeria-derived antigens to the

phagosomal cross-presentation pathway (see Sections 3.2.2 and 3.2.3)38.

Accordingly, electronic microscopy studies performed in macrophages

clearly established that not all bacteria gained access to the cytosol39.

Third, phagocytosis of dying infected cells by DCs may also deliver

Listeria-derived antigens to the phagosomal cross-presentation pathway39.

A recent report from Tvinnereim et al. supports the latter possibility (see

Section 3.5)40.

�55

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

Manipulation of bacterial pathways for antigen delivery to the cytosolic

pathway

The ectopic recombinant expression of the membranolytic LLO was

exploited to favor the cross presentation of antigens expressed by bacteria,

such as E. coli41 or M. bovis BCG42, that remain within vacuoles.

Vacuolar bacteria derived virulence factors are actively injected in the

cytosol to subvert host cell trafficking function. Russman et al. demonstrated

that genetic insertion of a model antigen into the Salmonella sptP gene,

which encodes a protein injected into host cell cytosol by a type III secretion

system, can lead to productive antigen presentation by a TAP-dependent

pathway43.

In addition, bacterial toxins endowed with intrinsic translocation

activities were used as a Trojan horse to deliver recombinant vaccinal T cell

epitopes in the cytosolic pathway of antigen presenting cells, including

DCs44.

3.2.2 The phagosomal pathway for cross presentation

A non-cytosolic pathway for vacuolar bacteria

Several bacteria restrict their growth to intracellular vacuoles. This is a way

to escape to bactericidal effector of humoral immunity such as antibodies

and complement. For a long time, it was thought that the MHC class I

pathway was restricted to cytosol-invading bacteria or viruses. Pioneering

studies from Harding, Pfeifer and Wick led to the discovery of an ‘‘alternative

pathway’’ for MHC class I loading in activated macrophages45. These

authors developed recombinant E. coli and Salmonella strains expressing

the chicken ovalbumin OVA model antigen. Infected macrophages

presented efficiently the immunodominant OVA peptide on MHC class I

molecules. Unlike the ‘‘classical’’ cytosolic pathway, this alternative pathway

was (i) resistant to proteasome inhibition, (ii) also observed in TAP deficient

macrophages, and (iii) insensitive to Golgi disruption by BFA treatment.

Phagolysosomal proteolysis and loading of antigenic peptides onto a post-

Golgi pool of MHC class I thus characterize this cross-presentation pathway.

However, all these experiments were performed in activated macrophages

raising the question of the relevance of this pathway in DCs. Recent

studies confirmed that this pathway was used for the cross presentation

of recombinant M. bovis BCG expressing the OVA antigen by both bone

marrow- and spleen-derived DCs46. In support of this, M. bovis BCG was

shown to act as an efficient vector for the delivery of antigens to the

phagosomal MHC class I pathway47–49.

�

56

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

The comparison of various types of particles and the decrease in antigen

presentation in TAP-deficient cells suggest that the phagosomal pathway

is of low efficiency and biased toward highly abundant phagosomal

antigens50,51. However, we note that the availability of MHC class I for

post-Golgi loading may be lower in TAP-deficient cells used to define this

pathway52–55. Indeed, in the absence of TAP, empty MHC class I molecules

accumulate in the ER. Therefore, the TAP-independent pathway may have

been underestimated in DCs due to this experimental limitation.

Post-Golgi MHC class I trafficking and loading

Initially, Pfeifer et al. showed that infected macrophages may support the

transfer of peptides to bystander antigen presenting cells45. This process

called ‘‘regurgitation’’ supports the idea that degradative macrophages

may cooperate in some way with DCs to generate bacteria-derived peptides.

However, there is yet no evidence of the existence of such a process in vivo.

Thus, the current view is that the phagosomal pathway involves MHC class I

loading in bacterial phagosomes or other vesicular post-Golgi compartments.

Do MHC class I traffic through the endocytic system in DCs?

Early studies demonstrated that MHC class I molecules surface level is

highly increased during DC maturation, due to a major increase in their

neosynthesis rate and to a modest stabilization of their half-life25–28. In

CD34-derived human Langerhans cells and in blood DCs, but not in

monocyte-derived DCs, MHC class I together with HLA-DM and HLA-DR

are found in late multivesicular MHC class II-containing endosomes56,57.

In maturing mouse DCs, MHC class I molecules transiently colocalize

with MHC class II and B7 molecules in non-lysosomal compartments,

probably trafficking to cell surface58. These endocytic population of

MHC class I may originate from the Golgi or from cell surface through

endocytosis. A recent paper from Jefferies’s laboratory addressed the role

of endosomal MHC class I trafficking by producing transgenic mice

expressing Kb molecules mutated on a conserved tyrosine in their cytosolic

domain59. Confirming previous studies showing that this tyrosine is

required for MHC class I endocytosis60,61, the endocytic localization of

tyrosine-mutated Kb molecules was reduced in immature transgenic DCs.

This result suggested that the contribution of Golgi-to-endosomes trafficking

to the MHC class I endosomal pool, if any, was limited. Strikingly, cross

presentation of exogenous proteins or viruses was markedly decreased in

these mice. Using a monoclonal antibody specific for OVA peptide-MHC

class I complexes, the authors showed that the tyrosine mutation affected

�57

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

MHC class I loading in endocytic positive compartments. Whether endo-

cytosis of MHC class I controls bacterial antigen cross presentation by DCs

remains to be determined.

The exchange of peptides already bound to MHC class I with

phagosome-derived peptides has been proposed as a mechanism for

endocytic MHC class I loading. However, phagosomes usually undergo

acidification and the consequence of a low pH environment on MHC class I

peptide exchange is not fully understood.

The pathway allowing MHC class I, once loaded, to reach cell surface

has not yet been well characterized in DCs but it is tempting to speculate

that it may share, at least in part, some transport pathways used by MHC

class II58 such as the tubulo-vesicular structures induced upon DC

maturation62–64.

3.2.3 Phagosomal antigens can access to the cytosolic pathway

As stated before, most of the pioneering studies using recombinant

bacteria expressing the OVA antigen were done in activated macrophages

with bacteria, such as E. coli or Salmonella, that do not actively disrupt their

vacuole. The development of DC culture protocols led to the reconsideration

of these issues in DCs using the same recombinant bacteria. Strikingly, the

cellular pathways used for cross presentation were proved to be different

in DCs compared to macrophages. Rescigno27 and Svensson65 identified

a ‘‘classical’’ cytosolic pathway for the cross presentation of vacuolar

OVA-expressing bacteria (Streptococcus gordonii, Salmonella). Indeed the

activation of OVA-specific CD8þ T cells was resistant to proteasome

inhibition and TAP-dependent. Accordingly, Salmonella infected DCs did

not exhibit the ability to ‘‘regurgitate’’ peptides to neighboring cells. These

results led to the conclusion that antigens from vacuolar bacteria gained

access to the cytosol in some way where they could meet proteasomal

degradation.

How exogenous antigens transit from bacterial vacuoles to the cytosol?

One may wonder whether a proportion of vacuolar bacteria may not

actually gain access to the cytosol and deliver antigens to the cytosolic

pathway. This hypothesis may be supported by reports of Salmonella66 or

Mycobacterium67,68 cytosol invasion. Moreover, the recent identification of

a repair system for cellular membranes injured by bacteria secretion

systems suggests that the maintenance of the vacuolar membrane integrity

is a dynamic and regulated phenomenon69. Supporting this view,

�

58

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

the SifA Salmonella protein was shown to be involved in the maintenance

of vacuole integrity70. Thus, even in the absence of global disruption,

transient permeability may be activated71.

Numerous studies support the existence of a cell transport process

linking phagosomal lumen to the cytosol. Pioneering studies from Rock’s

laboratory demonstrated in macrophages that phagocytosis of micron-sized

beads triggered specifically the egress of proteins from the phagosome to

the cytosol. Indeed, when beads are coated with the OVA antigen this leads

to its delivery to the cytosolic pathway, and when they are coated with the

cell impermeable gelonin toxin this leads to protein synthesis inhibition72.

Other groups have suggested that phagosome-to-cytosol pathway was

triggered by phagosome rupture due to the solid nature of latex beads73,74.

This was confirmed by the studies from Norbury et al. showing that growth

factor-induced macropinocytosis in macrophage75 and constitutive macro-

pinocytosis in immature DCs76 triggered the access of soluble proteins to

the cytosolic pathway. Later, it was shown that, in DCs, transport from the

endocytic pathway to the cytosol is selective for the size of the molecules

transported77. By contrast to antigens, endosomal resident proteins such

as active cathepsin D77 or L28 remain confined to the endocytic pathway.

This selectivity may rely on a specific sorting of exogenous protein anti-

gens or, more conceivably, to the non-specific release into the cytosol

of compartments that are not enriched with active cathepsin D or L. The

molecular machinery involved is still unknown but may require commu-

nication with the ER (see Section 3.2.4).

Of note, antigen transport from phagosomes to cytosol is more efficient

in DCs than in macrophages. This could explain why the same bacterial

antigen are presented by a TAP-dependent mechanism in DC and a TAP-

independent mechanism in macrophages77.

The TAP dependent MHC class I pathway for exogenous antigens is

highly regulated by a Toll-like receptor (TLR). First, TLR signalling promotes

rapidly the macropinocytic uptake of soluble antigens through the rapid

remobilization of intracellular actin pools from podosome to forming

ruffles78. Accordingly, Gil-Torregrossa et al. showed that DC pre-incubation

with LPS efficiently promotes the uptake of immune complexes and their

introduction into the cytosolic pathway24. Delamarre et al. also reported that

LPS promoted cross presentation of soluble antigen in early-activated DCs28.

The impact of TLR2–4 signalling on the dynamics of phagolysosomal

maturation has been studied in macrophages but it remains a controversial

issue79,80. How TLR signalling may influence cross presentation of bacteria

encoded antigens in DCs remains unknown.

�59

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

3.2.4 ER-phagosome fusion and cross presentation

ER-phagosome fusion defines a TAP-dependent ‘‘cross-presentation’’

compartment

As described in the previous section, most of the cross-presentation

pathways described in infected DCs were proteasome- and TAP-dependent

thus suggesting that ER was actually the compartment where MHC class I

loading takes place. However, morphological evidence directly supporting

this interpretation was slight. The characterization of ER recruitment to

phagosomal membranes challenged this view and led to the proposal of

a new model for cross presentation.

A pioneering paper from Desjardin’s lab established that phagocytosis

of inert as well as bacterial particulate substrate actually involved ER-

phagosome fusion. Gagnon et al. performed morphologic analysis of latex

beads containing phagosomes in murine macrophages using electronic

microscopy81. Phagosomal membranes contain numerous domains positive

for glucose-6-phosphatase or calnexin, two ER markers. Moreover, proteomic

analysis of fractionated phagosomes revealed that they contain numerous

ER proteins82. Morphological analysis of phagocytic cups suggested that

ER recruitment may occur by direct fusion between the ER and the plasma

membrane81. The same observations were obtained in DCs83,84.

What is the impact of ER recruitment to the proteasome- and

TAP-dependent pathway?

First, ER-derived membranes may provide a machinery for the export

of phagosomal antigens to the cytosolic proteasome-dependent pathway.

Indeed, numerous misfolded proteins of the ER lumen are exported to the

cytosol, where they are deglycosylated, ubiquitinylated and finally targeted

to proteasome degradation, a process called ER-associated degradation

(ERAD) (for a review see Ref. 85). Importantly, ERAD can promote the

presentation of lumenal proteins from ER by a proteasome and TAP-

dependent pathway. ERAD has been coopted by viruses to trigger MHC

class I breakdown86. In this case, co-precipitation experiments suggest

that Sec61 may act as transporter for delivering ERAD substrates to the

cytosol87 and possibly some bacterial toxins whose translocation to the

cytosol starts in the ER85. However, other studies indicate that the US2/11-

mediated retrotranslocation may bypass protein unfolding, questioning

the contribution of Sec61 whose substrates may be limited to fully

unfolded proteins88. Recent results from the groups of H. Ploegh89 and

T. Rappoport90, implicate the membrane protein Der1p in the degradation

�

60

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

of MHC class I heavy chain triggered by US11. Der1p associates with both

US11 and with VIMP, a novel membrane protein that recruits the cytosolic

p97 AAA ATPase required for MHC class I retro-translocation and

proteasome targeting of ER-derived substrates90,91, Phagosome-associated

proteasomes may be involved in the degradation of phagosomal antigens

targeted to the cytosol92, The contribution of these pathways to cross

presentation remains to be established.

Second, the ER-phagosome fusion model led to the hypothesis that

phagosomes may represent competent organelles for TAP-dependent

loading of proteasome derived peptides. This hypothesis received strong

experimental support. Two studies showed that purified phagosomes

indeed support ATP-dependent TAP-mediated peptide import83,84.

Moreover, phagosomal MHC class I is integrated in a loading complex

encompassing chaperones and oxydoreductases that participate in the

assembly of peptide-MHC complexes (calreticulin, Erp57 and tapasin),

which are physically linked to TAP19,84. The glycosylation status of the

phagosomal MHC class I associated to TAP confirms that it is mostly from

pre-Golgi origin84. Moreover it could be hypothesized that MHC class I

trafficking from cell surface through its internalization motif may also

associate with loading complexes from ER origin59. Whatever its origin,

in vitro experiments demonstrated that phagosomal MHC class I is

accessible to cytosolic peptides imported by phagosomal TAP transporters83.

MHC class I molecules loaded with a peptide derived from phagocytosed

antigen were detected in phagosomes, using both antigen-specific T cells

and a monoclonal antibody specific for the same complex83. As for cell

surface appearance of MHC class I-peptide complexes, phagosomal peptide-

MHC class I complexes were not detected when the cells were treated with

proteasome inhibitors, indicating that peptide generation occurred in the

cytosol. The contribution of this loading pathway, as compared to ER

loading, to cross presentation remains unclear. MHC class I-peptide

complexes were found to be enriched in antigen-bearing phagosomes, as

compared to control phagosomes in the same cells83. These results suggest

that MHC class I loading is favored in antigen-bearing phagosomes,

as compared to other compartments such as ER or phagosomes that do

not contain antigen.

How do peptide-MHC class I complexes loaded in phago-ER-some reach

cell surface?

MHC class I may travel through the Golgi or directly to cell surface via

an endocytic recycling route. The partial inhibition observed with the

�61

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

Golgi-disrupting agent BFA suggest that the second alternative is possible92.

In support of this, Ackerman et al. observed that a portion of cell surface

MHC class I retains a pre-Golgi glycosylation status, suggesting that it

bypassed Golgi trafficking25,84.

The ER-phagosome model give an explanation for the high efficiency

of phagocytic cross presentation. However, it does not explain how

soluble antigens acquired by constitutive macropinocytosis could access

TAP-dependent MHC class I presentation76. Elegant studies from

Cresswell’s laboratory have recently answered this question. Ackerman

et al. showed that the cytomegalovirus soluble protein US6, known to

inhibit TAP function by interacting with its luminal domain, inhibited

the cross presentation of soluble OVA84. These data demonstrated that

soluble antigens have access in some way to compartment presenting

ER components. Strikingly, US6 also inhibited significantly the TAP-

dependent presentation of endogenous proteins93. Moreover, exogenous

b2-microglobulin localized rapidly to the perinuclear ER, associated with

MHC class I heavy chains, and rescued both cell surface class I expression

in b2 microglobulin �/� cells and presentation of endogenous antigens93.

These data give evidence that soluble antigens can access most of ER

lumen in DCs where they gain access to the cytosolic pathway.

ER recruitment to bacterial vacuoles: a role in cross presentation?

Legionella pneumophila and Brucella abortus are able to create replicative

organelles within macrophages, by actively inducing ER recruitment to the

bacterial phagosome. The control of ER-phagosome biogenesis depends in

both cases on a functional type IV secretion system that injects virulence

factors in the host cell cytosol (the Dot/Icm system for Legionella94 and the

VirB system for Brucella)95,96. Even though their ER-derived replicative

vacuoles present morphological similarities, Legionella and Brucella trigger

active ER-phagosome fusion through distinct pathways.

Legionella avoid fusion with early endosomes and acquires ER

membranes through the subversion of ER to Golgi vesicular trafficking.

As shown by Roy’s lab, Arf1 is activated by the Dot/Icm-injected RalF

guanine nucleotide exchange factor that participates in its recruitment

to Legionella phagosomes97. There, Arf1 plays a crucial role in sustained

bacterial growth. The small GTPase Rab1 and the SNARE Sec22, are

required for the subversion of the formation of Legionella replicating

vacuole98. Interestingly, yeast Sec22 was shown to support the fusion

of liposomes with plasmalemma-derived SNAREs, thus suggesting that

it may regulate ER-phagosome fusion99. This idea recently received

�

62

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

experimental evidence in mammalian cells: the phagocytose of 3 mm latex

beads by J774 macrophages is blocked by microinjection of anti-Sec22b

antibodies or expression of dominant negative forms of Sec22b100.

After the acquisition of certain lysosomal markers95, Brucella

containing-vacuoles intercept the host secretory pathway at a slightly earlier

point as compared to Legionella by a process that requires the activity of

the Sar-1 small GTPase95. The relevance of ER-derived membrane acquisi-

tion for cross presentation of Brucella or Legionella antigens remains to

be tested.

3.3 MHC CLASS II PATHWAYS

3.3.1 The endo-lysosomal pathway for MHC class II presentation

The formation of peptide-MHC class II complexes for presentation to CD4þ

T cells is an endocytic process associated to endosomal proteolysis both at

the level of antigen processing and of MHC class II maturation pathway.

Here, we will only summarize briefly tremendous work that has been

nicely reviewed elsewhere101,102.

Neosynthesized MHC class II a and b chains assemble in the

endoplasmic reticulum (ER) with a non-polymorphic transmembrane

protein called invariant chain (Ii). Ii triggers the formation of nonameric

complexes encompassing three MHC class II dimmers and three Ii

molecules. A cytoplasmic domain of Ii addresses these complexes from the

ER to the endocytic pathway through the Trans-Golgi Network. A minor

portion of neosynthesized MHC class II gain direct access to the cell

surface before being internalized into endosomes. Successful antigen

presentation relies on breakdown and removal of Ii, which must be

proteolyzed upon arrival to endosomes to free the class II-peptide binding

groove for exchange with the antigenic peptide. Peptide exchange is catalyzed

by the chaperone H2-DM whose activity is triggered at acidic pH. In addition,

the last step of Ii proteolysis liberates MHC class II from the cytosolic tail

of Ii, which contains the endosomal retention motif and thereby allows

MHC class II-peptide complexes to reach the cell surface. This step of

Ii degradation is under the control of the cysteine protease, cathepsin S,

in both DCs and B lymphocytes. Antigenic peptides arise from the com-

bined action of various endocytic proteases. The degradation task of these

enzymes can eventually be facilitated by the acid-activated oxido-reductase,

GILT, which contributes to antigen unfolding by breaking di-sulfide bonds.

�63

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

Most of these hydrolases display optimal activity under the acidic conditions

that are characteristic of late-endosomal/lysosomal compartments.

In DCs, the MHC class II pathway is submitted to a tight developmental

regulation. In association with their tissue localization, immature DCs

exhibit a high ability to capture antigens by various endocytic processes

such as phagocytosis, macropinocytosis or receptor-mediated endocytosis.

Immature DCs may form special endocytic retention compartments

devoid of V-ATPase thus retaining internalized antigens intact for further

processing in mature DCs103. Protease inhibitors may also dampen

proteolytic activity in immature DCs104. Immature DCs display a reduced

lysosomal acidification, associated with a limited antigen proteolysis

and Ii cleavage. Cell surface MHC class II are also efficiently endocytozed102.

As a result, MHC class II are mostly intracellular and the ability of

immature DCs to process antigens is low, especially for the most stable

proteins.

Upon induction of maturation by LPS, DCs undergo a coordinated set

of modifications associated with their migration toward lymphoid organs.

One major event is the acidification of late endocytic compartment

and activation of lysosomal function, promoting both antigen and Ii

processing105. This event is triggered by the recruitment and docking of

the V-ATPase-cytosolic subunit V1 to the lysosomal membrane-associated

V0 subunit, but the molecular mechanisms regulating this event remain

to be established105. At the level of trafficking, MHC class II are targeted

toward tubular compartments linking the late endocytic pathway to cell

surface62. In mature DCs, the endocytosis of cell surface MHC class II is

deeply inhibited and as a result, high levels of MHC class II accumulate at

the cell surface106. Moreover, in mature DCs, the half life of MHC class II

is greatly increased thus sustaining prolonged antigen presentation to

CD4þ T cells in the lymph nodes. Thanks to all these coordinated

modifications, once matured, DCs display the striking ability to present

antigens, even several days after endocytic uptake, a phenomenon termed

as ‘‘antigenic memory’’ (reviewed in Ref. 101). In conclusion, developmental

regulation of DC function allows the precise coordination of antigen

transport and degradation in the course of DC migration from peripheral

organs where they capture antigens, to lymphoid organs where they present

antigens to T cells101,107.

Important progress in the characterization of phagosome function

in MHC class II presentation was performed in macrophages. Ramachandra

et al. showed that phagosomes carrying inert beads matured toward

phagolysosomes and acquired MHC class II, Ii and DM. Nascent

�

64

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

MHC class II trafficking from Golgi and recycling MHC class II derived from

cell surface were found in phagosomes. These phagosomes are degra-

dative organelles where antigen and Ii are efficiently processed108,109.

Thus, they have all the features of a MHC class II loading compartment.

However, this does not exclude that loading may take place in other late

endocytic compartments upon antigen or peptide vesicular transport.

To resolve this issue, Ramachandra et al. developed a unique experimental

approach to identify specific MHC class II-peptide in the membrane of

fractionated organelles. Using this technique, the authors demonstrated

that phagosomes were competent for MHC class II loading of peptides

derived from phagocytozed antigens. MHC class II loading was not

detectable in non-phagosomal organelles, showing that antigen (or peptide)

export to other organelles was not a significant phenomenon, at least

for MHC class II loading. Phagosomal MHC class II loading pathway use

primarily nascent MHC class II derived from the Golgi108,109. The same

authors applied this approach in order to analyze the MHC class II-

restricted presentation of a peptide derived from the Ag85B secreted by

Mycobacterium tuberculosis (Mtb)110. This study formally established that

the Mtb-containing phagosome is actually the major organelle for specific

MHC class II loading in activated macrophages even if some escape

mechanisms are set up by live bacteria to inhibit phagolysosomal maturation

and the MHC class II pathway (see Section 3.4)110.

3.3.2 Cytosol derived antigens can access the MHC class II

pathway

A strong CD4 T cell response has been reported following mice infection

with the intracellular bacteria Listeria monocytogenes. Moreover, macrophages

and DCs isolated from these mice efficiently present Listeria-derived

epitopes on MHC class II111,112. Therefore, despite the intracytosolic loca-

tion of Listeria, its secreted proteins have a direct access to the endocytic

MHC class II presentation pathway. A possibility would be that DCs acquire

bacterial-derived antigen by phagocytosis of dead bacteria or of Listeria-

infected cells. In vitro experiments by Skoberne et al. validated this

hypothesis, but also showed that the direct infection of DCs and macro-

phages can lead to the presentation of Listeria-derived epitopes on MHC-II

molecules113.

The fact that bacterial cytosolic antigens can be presented on MHC

class II is not so surprising. Indeed, a large proportion of peptides bound

to MHC class II, derived from endogenous proteins residing in the cytosol

�65

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

(reviewed in Ref. 101). However, the underlying mechanisms allowing

cytosolic antigens access to a compartment competent for loading on

MHC class II are poorly characterized at the cell biology level.

Despite exceptions114, it seems that this pathway is independent of

the proteasome and of the ER resident peptide transporter TAP. Since the

process of peptide loading on MHC class II (Ii release, DM activation)

require endosomal acidification, the access to endocytic pathway is more

likely to be required. Indeed, the direct transport of peptides derived from

cytosolic proteins into late endosomal compartment positives for LAMP-1

and cathepsin-D was reported115.

But how is the connection between the exocytic and the endocytic

pathways achieved?

A system called chaperone-mediated autophagy, activated upon metabolic

stress, triggers the transfer across membranes of cytosolic proteins contain-

ing a particular targeting sequence, directly into endosomes and lyso-

somes116. Interestingly, the two major constituents of this system,

hsc70 and Lamp2a, were recently reported to facilitate MHC class II

presentation of cytosolic antigens117. However, there is no evidence that

cytosolic bacterial antigens can be transported into lysosomes by this process.

Another cellular pathway called macroautophagy is involved in cytosolic

protein turn-over and is more likely to be involved in MHC class II

loading of cytosolic antigens. Indeed, this process, induced in response

to cell starvation, triggers the sequestration of cytosolic components into

double-membrane bound vacuoles called autophagosomes for later degrada-

tion in lysosomal compartments.

Recent studies point out a link between autophagy and the delivery

of cytosolic peptides for MHC-II-restricted presentation. Indeed, Paludan

et al. have shown that silencing of Apg12, an essential autophagy gene,

by RNA interference, inhibits MHC class II-restricted presentation of the

Epstein–Barr virus antigen EBNA1118. Moreover, Dengiel et al. have reported

that induction of autophagy strongly enhances MHC-II presentation of

peptides derived from cytosolic proteins119.

Insights in the role of the autophagic pathway were made possible

thanks to the identification of specific markers of mammalian autophagy

(Apg5, Apg12 and LC3)120. Interestingly, cell biology studies exploiting

these new tools revealed a link between the autophagic pathway and

intracytosolic pathogenic bacteria behavior in infected cells. Indeed,

autophagy appears to be a bactericidal pathway controlling the targeting

�

66

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

of cytosolic bacteria into lysosomal compartments. As an example, the

pathogenic group A Streptococcus is destroyed by the autophagic machinery

in human cells36. In contrast, Shigella flexneri and Listeria monocytogenes

prevent this process through the action of virulence effectors37,38.

Therefore, the autophagic process might be a way to bring cytosolic bac-

terial antigens to MHC class II for loading into endocytic compartments,

resulting in MHC class II presentation of bacterial epitopes at the cell

surface that elicit CD4 T cell responses against pathogens.

3.4 THE INTERPLAY BETWEEN BACTERIAL VIRULENCE AND

MHC CLASS I AND CLASS II PATHWAYS

DCs are permissive to infection by low virulent bacterias

MacPherson et al. established that gut-derived DCs, but no other cell types,

carry live commensal bacteria to mesenteric draining lymph nodes after their

uptake in the gut121. Once in lymph nodes, DC can interact directly with

B cells to promote their differentiation in IgA secreting cells121. However,

even if IgA production to commensals seems to bypass antigen presenta-

tion to T cells122, one may speculate that the transport of live bacteria to

draining lymph nodes could promote antigen presentation by MHC class I

or II to T cells. Nevertheless, the elegant studies from MacPherson et al.

strongly suggest that DCs are endowed with specific mechanisms allowing

them to maintain phagocytozed bacteria alive even after the completion of

the migration phase from tissues to draining lymph nodes121.

The low bactericidal activity of DCs toward commensals is coherent

with their ability to support infection by bacterial mutants devoid of

essential virulence factors and unable to infect macrophages. Indeed,

Niedergang and Garcia del Portillo have shown that DCs, unlike macro-

phages, were efficiently infected by PhoP-deficient Salmonella strains9,123.

The PhoP/PhoQ system controls the coordinated expression of virulence

genes involved in bacterial invasion and intracellular survival. Moreover,

DCs presented Salmonella-derived antigens on MHC class II independently

of the PhoP-regulated virulence factors9. The high macropinocytic activity

of immature DCs promotes Salmonella entry independently of the Salmonella

pathogenicity island (SPI)-1 type III secretion system absent in PhoP

mutant strain. This suggests that the Salmonella-containing vacuoles differ

in DCs and macrophages. Indeed, the lack of lysosomal glycoproteins

acquisition by Salmonella vacuoles in DCs supports this view123. This

underscores how DC and macrophage endocytic pathways may face

�67

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

bacteria to totally different constraints, probably in relation to the reduced

lysosomal function of DCs (at least in the immature stage).

Bacterial inhibition of phagolysosomal function and antigen presentation

in macrophages and DCs

Uptake of inert particles such as latex beads leads to the formation of

a phagosome inside the host cell. Upon multiple fusion and fission events

with other vesicular organelles, this phagosome progressively becomes

a phagolysosome, a process called phagosomal maturation124. Pathogenic

vacuolar bacteria invade cells through a phagocytic process and actively

inhibit phagolysosome formation in order to create a niche for their

replication.

For example, Mycobacterium tuberculosis (Mtb) actively inhibits the

delivery of V-ATPase to its containing-vacuole125 and prevents phagolyso-

somal maturation126. Deretic’s laboratory had a central contribution to

understand this process in which phosphoinositol-3-phosphate (PI3P)

metabolism play a central role. Indeed, PI3P formation is essential for

phagolysosome biogenesis as it recruits EEA1 which cooperates with

syntaxin6 in the delivery of lysosomal hydrolases and V0-ATPase from

the Trans-Golgi Network to phagosomes127. Mtb cell wall glycolipid

lipoarabinomannan (LAM) inhibits the activation of the PI3-kinase

hVPS34 by blocking a Ca2þ-activated calmodulin/CaMKII signalling

cascade128,129. Moreover live Mtb secretes a phosphatase called SapM that

inhibit phagolysosomal maturation by hydrolyzing PI3P130. As PI3P also

recruits the p40 subunit of NADPH-oxidase which positively regulates

superoxide production, the inhibition of PI3P formation at the membrane

of live Mtb-containing phagosomes, could also have an effect in diminish-

ing the oxidative burst131. The inhibition of phagolysosomal function by

live Mtb is associated with an inhibition of MHC class II presentation that

was mostly studied in monocytes/macrophages (reviewed in Ref. 132).

Ramachandra et al.133 demonstrated that inhibition of MHC class II

presentation involve factors resistant to certain procedure of bacterial

killing except heat-killing (possibly LAM, or TLR2 agonists, see below).

The inhibition by Mtb of PI3P-dependent trafficking from Trans-

Golgi Network to Mtb vacuole, may also concern the recruitment of

neosynthesized MHC class II and DM molecules toward Mtb vacuole134.

Accordingly, Ii processing and peptide loading on MHC class II where

inhibited as demonstrated biochemically by the reduction of SDS-stable

peptide-loaded MHC-IIa/b dimmers in Mtb-infected cells134. This study

points out the link between subvertion of cell transport pathways by Mtb

�

68

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

and the inhibition of MHC class II presentation. However, the picture may

be more complex as other inhibitory mechanisms seem to directly target

the IFNg-induced CIITA transactivator of the genes coding for MHC class II,

Ii and DM. The activation of TLR2 by the Mtb 19 kD lipoprotein controls

in macrophages the inhibition of transcriptional activity by mechanisms

that remains to be determined (reviewed in Ref. 132). Of note, MHC class I

surface levels seem to be less affected upon Mtb infection, perhaps in relation

with the protective role of CD8þ T cells in chronic infection135.

A likewise major consequence of the inhibition of antigen presentation

in infected macrophages is that the delivery of bacteriostatic cytokines

provided by CD4þ T cells is inhibited. However, the relevance of the

phagolysosomal inhibition for the priming of CD4þ (and CD8þ) T cells

by Mtb-infected DCs remains to be carefully analyzed.

DC infection by Salmonella typhimurium was reported to inhibit the

MHC class II presentation of bystander antigens through a mechanism

dependent of the SPI-2 type III secretion system. Accordingly, SPI-2 coding

genes expression is actually induced in Salmonella infected DCs but the

SPI-2 system is dispensable for DC infection136,137. Interestingly, Salmonella-

mediated inhibition of MHC class II pathways can be overcome by FcgR-

mediated internalization of opsonized bacteria as a consequence of bacteria

targeting to phagolysosomal degradation138. These reports establish that

MHC presentation pathways in DCs can be a target for bacterial escape.

Constrained survival of virulent bacteria in DCs and the presentation

of bacterial antigens

Several studies established that infected DCs can restrict the growth of

intracellular bacteria without killing them. Using a segregative plasmid,

Jantsch et al. elegantly demonstrated that Salmonella infecting DCs

represented a live, non-dividing population136. Jiao et al. demonstrated that

early after M. bovis BCG infection, splenic DCs efficiently present M. bovis

BCG secreted antigens7. Interestingly, bacilli survive and remain stable in

DCs up to two weeks after infection. These studies suggest that antigen

presentation – at least for secreted antigens – may take place in chronically

infected DCs carrying live bacilli. Moreover, infection of mice with a

recombinant M. bovis BCG strain expressing the OVA antigen supports

a long-lasting antigen presentation in vivo48,49.

In vitro studies from Neyrolles’s group promote the idea that

human DCs do not behave as macrophages with respect to mycobacterial

infection: Tailleux et al. showed that DCs restrict the growth of Mtb. In DCs,

like in macrophages, live Mtb-containing phagosomes do not mature to

�69

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

phagolysosomes139. However, unlike in macrophages, the Mtb phagosome

is disconnected from the early recycling pathway and this may restrict the

accessibility of bacilli to exogenous nutrients such as iron or cholesterol139.

Whether antigens derived from this vacuole may enter some presentation

pathway remains to be determined. As proposed by Tailleux et al., the distinct

intracellular route of Mtb phagosome in DCs and in macrophages could

result from the engagement of different receptors during phagocytosis:

Mtb uptake by human DCs, but not by macrophages, is mediated by an

interaction between Mtb LAM and the DC lectin-surface receptor DC-SIGN6.

Legionella pneumophila inhibits the phagolysosomal maturation of its

vacuole and actively promotes the formation of an ER-containing replicating

vacuole. Studies from the Roy lab established that DCs but not macro-

phages restrict the growth of Legionella140. Legionella is able to prevent

fusion of phagosome with lysosomes in both cell types through the action

of the Dot/Icm system. Microscopy studies performed by Neild et al. in

Legionella-infected DCs, demonstrated that phagosomes acquired ER

membranes in a manner similar to what have been shown in macrophages.

Moreover, despite a restricted growth, Legionella remains metabolically

active and retain their ability to neosynthesize proteins as shown by the

induction of green fluorescent protein (GFP) under an inducible pro-

moter140. Strikingly, the constriction of bacterial growth does not exclude

neosynthesized bacterial antigens from the class II presentation pathway.

Characterization of the transport pathways allowing the presentation

of the neosynthesized bacterial antigens is of major interest. These

pathways may be specially relevant for the induction of T cell immunity

against secreted bacterial antigens by infected DCs carrying live bacteria

during their migration to the T cell zones of lymphoid organs.

However, T cell responses against non-secreted bacterial antigens

may rely on the intracellular killing and degradation of bacteria. Therefore,

it would be interesting to determine whether the developmental regulation

of phagolysosomal function that has been characterized for model

antigens101 is also relevant for bactericidal functions. In this case, one

may wonder whether DCs may kill and process bacteria once they reached

the T cell zones of lymphoid organs.

3.5 BACTERIA-INDUCED APOPTOSIS AND CROSS

PRESENTATION OF BACTERIAL ANTIGENS

Bevan et al. discovered that cell-associated antigens of engrafted cells

could be presented on the MHC of host antigen presenting cells141.

�

70

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

This process was called cross priming. Numerous in vitro and in vivo studies

gave some cellular basis to this process: antigen presenting cells can acquire

and process cell-associated antigens from apoptotic bodies they phago-

cytose142. As reviewed elsewhere, this process may be a major relevance for

the induction of antiviral T cell responses143. Bacterial infection can trigger

apoptosis in many cell types. This led to the suggestion that the phagocytosis

of dead cells harboring bacterial antigens may participate to the induction

of anti-bacterial adaptative immune responses through cross presentation.

This idea received experimental first evidence in vitro in a study from

Wick’s laboratory13. Apoptotic bodies of Salmonella infected macrophages

were efficiently phagocytozed by DCs triggering the cross presentation

of Salmonella encoding antigens by MHC class I of the phagocytic DCs.

In this model, cross presentation of bacteria infected apoptotic

bodies seems to follow a ‘‘classical’’, cytosolic, pathway involving antigen

access to the cytosol, proteasomal degradation and TAP-dependent loading13.

This is in line with what has been characterized for cell-associated viral

antigens144–146. The contribution of ER-mediated phagocytosis to the

phagocytic uptake of dead cells and the transport pathways linking

phagosome lumen to cytosol remain key issues that have not yet been

documented. Several groups implicated long-lived proteins from dead

cells as the antigenic substrate transported from dead cell bodies to cross

presenting DCs147–149. Other groups sustain that HSP-mediated transfer

of antigenic peptides from the dead cell to the cross presenting

DCs150–152. Interestingly, bacteria-derived HSPs may also shuttle bacterial

antigenic peptides to cross-presenting DCs153.

Schaible et al. demonstrated that Mtb-triggered apoptosis produced

some blebs harboring proteic and lipidic antigens that were efficiently

uptaken and cross presented by bystander DCs on classical as well as

CD1b MHC molecules154. However, the existence of this pathway does not

exclude direct presentation by chronically infected DCs.

In vivo physiological relevance of cross presentation after uptake of

dead cell carrying bacterial antigens remains ill defined.

In the case of Salmonella, PhoP-deficient strains do not induce

either macrophage apoptosis or cross presentation in vitro13. In vivo,

Wijburg et al. demonstrated that SPI-1 (controlled by PhoP) deficient strains

still induce CD8þ T cell activation for bacterial antigens155. This suggests

that direct infection of DCs may be the main route for CD8þ T cell priming

against Salmonella antigens. However, other SPI-1-independent apoptotic

pathways may provide dead cells harboring Salmonella antigens to cross

presenting, phagocytic DCs.

�71

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S -PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

In the case of the Listeria infection, indirect arguments suggest that

the dead cell pathway may be of major relevance. Indeed, virulence

factors trigger the rapid apoptosis of infected DCs both in vitro and

in vivo29. Using adoptive transfer experiments to monitor antigen presenta-

tion in vivo, Wong et al. elegantly demonstrated that CD8þ T cells are

stimulated during the first days of infection156. However, the quick

disappearance of antigen presenting cells is not only due to Listeria-

induced apoptosis since activated CD8þ T cells also participate in the

process156. Therefore, these studies do not rule out either direct infection

or cross presentation as main priming mechanisms. Geginat’s laboratory

successfully modelized in vitro the cross presentation of Listeria derived

epitopes by both MHC class I and II113,157. Interestingly, the process may

be of special relevance for MHC class II presentation of Listeria-derived

epitopes that could not gain efficiently access to phagolysosomes otherwise

(see Section 3.3.2). Interestingly, neutrophils may play a role in this

process by providing numerous infected dead cells carrying listerial antigens

to cross-presenting DCs. In support of this hypothesis, Tvinnereim et al.

have shown that dead neutrophils serve as efficient substrates for cross

presentation both in vitro and in vivo40. The relevance of this pathway

is demonstrated by in vivo experiments in which neutrophil depletion

deeply inhibits the priming of CD8þ T cells specific for non-secreted

antigens40. Strikingly, the priming of T cells specific for secreted antigens

is not affected in these conditions. Although indirect, these data argue

for a major role of cross presentation at least in the case of non-secreted

listerial antigens.

3.6 CONCLUSION: BACTERIAL COMPARTMENTALIZATION AND

MHC PRESENTATION PATHWAYS

Historically, the MHC-II pathways were primarily thought to be more or

less restricted to endocytically acquired antigens whereas the MHC class I

was believed to be assigned to the presentation of cytosol-derived antigens.

Cell biology studies clearly demonstrated that this dichotomy is no more

relevant in face of the complexity and variety of cellular transport pathways.

At the theoretical level, it can be postulated that every bacteria, indepen-

dently of its intracellular location, may provide antigens presented by MHC

class I and class II pathways. This is of outstanding interest given the

cooperation of CD8þ and CD4þ T cells in mounting efficient protective

anti-bacterial memory T cell responses. However, the precise interplay

between bacterial virulence factors, host anti-bacterial pathways and

�

72

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

MHC antigen presentation pathways remains to be better defined in

physiologically-relevant DC subsets.

ACKNOWLEDGEMENTS

L. B.-V. was supported by the Association pour la Recherche contre le Cancer,

and P. G. was supported by the CNRS. The authors thank Ana-Maria

Lennon-Dumenil and Stephanie Hugues for critical reading of the manu-

script. Due to space limitation we could only cite a fraction of the published

work, which does not undermine the great value of uncited studies.

REFERENCES

1. Banchereau, J. et al. (1998). Nature 392(6673), 245–52.

2. Guermonprez, P. et al. (2002). Ann. Rev. Immunol. 20, 620–67.

3. Kolb-Maurer, A. et al. (2000). Infect Immun. 68(6), 3680–8.

4. Pron, B. et al. (2001). Cell Microbiol. 3(5), 331–40.

5. Bodnar, K. A. et al. (2001). Infect. Immun. 69(2), 800–9.

6. Tailleux, L. et al. (2003). J. Exp. Med. 197(1), 121–7.

7. Jiao, X. et al. (2002). J. Immunol. 168(3), 1294–301.

8. Hopkins, S. A. et al. (2000). Cell Microbiol. 2(1), 59–68.

9. Niedergang, F. et al. (2000). Proc. Natl Acad. Sci. U S A 97(26), 14650–5.

10. Yrlid, U. et al. (2002). J. Immunol. 169(1), 108–16.

11. Svensson, M. et al. (2000). Infect. Immun. 68(11), 6311–20.

12. Johansson, C. et al. (2004). J. Immunol. 172(4), 2496–503.

13. Yrlid, B. U. et al. (2000). J. Exp. Med. 191(4), 613–24.

14. Lenz, L. L. et al. (2000). J. Exp. Med. 192(8), 1135–42.

15. Jung, S. et al. (2002). Immunity 17(2), 211–20.

16. Yewdell, J. W. et al. (2003). Nat. Rev. Immunol. 3(12), 952–61.

17. Rock, K. L. et al. (2004). Nat. Immunol. 5(7), 670–7.

18. Kloetzel, P. M. (2004). Nat. Immunol. 5(7), 661–9.

19. Cresswell, P. (2000). Traffic 1(4), 301–5.

20. Macagno, A. et al. (1999). Eur. J. Immunol. 29(12), 4037–42.

21. Lelouard, H. et al. (2002). Nature 417(6885), 177–82.

22. Lelouard, H. et al. (2004). J. Cell Biol. 164(5), 667–75.

23. Canadien, V. et al. (2005). J. Immunol. 174(5), 2471–5.

24. Gil-Torregrosa, B. C. et al. (2004). Eur. J. Immunol. 34(2), 398–407.

25. Ackerman, A. L. et al. (2003). J. Immunol. 170(8), 4178–88.

26. Cella, M. et al. (1999). J. Exp. Med. 189(5), 821–9.

27. Rescigno, M. et al. (1998). Proc. Natl Acad. Sci. U S A 95(9), 5229–34.

�73

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

28. Delamarre, L. et al. (2003). J. Exp. Med. 198(1), 111–22.

29. Pamer, E. G. (2004). Nat. Rev. Immunol. 4(10), 812–23.

30. Pamer, E. G. et al. (1997). Immunol. Rev. 158, 129–36.

31. Villanueva, M. S. et al. (1994). Immunity 1(6), 479–89.

32. Villanueva, M. S. et al. (1995). J. Immunol. 155(11), 5227–33.

33. Princiotta, M. F. et al. (2003). Immunity 18(3), 343–54.

34. Sijts, A. J. et al. (1997). J. Biol. Chem. 272(31), 19261–8.

35. Shen, H. et al. (1998). Cell 92(4), 535–45.

36. Nakagawa, I. et al. (2004). Science 306(5698), 1037–40.

37. Ogawa, M. et al. (2005). Science 307(5710), 727–31.

38. Rich, K. A. et al. (2003). Cell Microbiol. 5(7), 455–68.

39. Portnoy, D. A. et al. (1989). J. Exp. Med. 170(6), 2141–6.

40. Tvinnereim, A. R. et al. (2004). J. Immunol. 173(3), 1994–2002.

41. Higgins, D. E. et al. (1999). Mol. Microbiol. 31(6), 1631–41.

42. Hess, J. et al. (1998). Proc. Natl Acad. Sci. U S A 95(9), 5299–304.

43. Russmann, H. et al. (1998). Science 281(5376), 565–8.

44. Guermonprez, P. et al. (1999). J. Immunol. 162(4), 1910–16.

45. Pfeifer, J. D. et al. (1993). Nature 361(6410), 359–62.

46. Cheadle, E. J. et al. (2005). Infect. Immun. 73(2), 784–94.

47. Winter, N. et al. (1995). Vaccine 13(5), 471–8.

48. van Faassen, H. et al. (2004). J. Immunol. 172(6), 3491–500.

49. Dudani, R. et al. (2002). J. Immunol. 168(11), 5737–45.

50. Wick, M. J. et al. (1996). Eur. J. Immunol. 26(11), 2790–9.

51. Shen, L. et al. (2004). Immunity 21(2), 155–65.

52. Harding, C. V. et al. (1994). J. Immunol. 153(11), 4925–33.

53. Song, R. et al. (1996). J. Immunol. 156(11), 4182–90.

54. Chefalo, P. J. et al. (2003). J. Immunol. 170(12), 5825–33.

55. Chefalo, P. J. et al. (2001). J. Immunol. 167(3), 1274–82.

56. MacAry, P. A. et al. (2001). Proc. Natl Acad. Sci. U S A 98(7), 3982–7.

57. Kleijmeer, M. J. et al. (2001). Traffic 2(2), 124–37.

58. Turley, S. J. et al. (2000). Science 288(5465), 522–7.

59. Lizee, G. et al. (2003). Nat. Immunol. 4(11), 1065–73.

60. McCluskey, J. et al. (1986). EMBO J. 5(10), 2477–83.

61. Vega, M. A. et al. (1989). Proc. Natl Acad. Sci. U S A 86(8), 2688–92.

62. Kleijmeer, M. et al. (2001). J. Cell Biol. 155(1), 53–63.

63. Chow, A. et al. (2002). Nature 418(6901), 988–94.

64. Boes, M. et al. (2002). Nature 418(6901), 983–8.

65. Svensson, M. et al. (1999). Eur. J. Immunol. 29(1), 180–8.

66. Brumell, J. H. et al. (2002). Curr. Biol. 12(1), R15–17.

67. Stamm, L. M. et al. (2003). J. Exp. Med. 198(9), 1361–8.

�

74

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

68. McDonough, K. A. et al. (1993). Infect. Immun. 61(7), 2763–73.

69. Roy, D. et al. (2004). Science 304(5676), 1515–18.

70. Beuzon, C. R. et al. (2000). EMBO. J. 19(13), 3235–49.

71. Teitelbaum, R. et al. (1999). Proc. Natl Acad. Sci. U S A 96(26), 15190–5.

72. Kovacsovics-Bankowski, M. et al. (1995). Science 267(5195), 243–6.

73. Reis e Sousa, C. et al. (1995). J. Exp. Med. 182(3), 841–51.

74. Oh, Y. K. et al. (1997). Vaccine 15(5), 511–18.

75. Norbury, C. C. et al. (1995). Immunity 3(6), 783–91.

76. Norbury, C. C. et al. (1997). Eur. J. Immunol. 27(1), 280–8.

77. Rodriguez, A. et al. (1999). Nat. Cell Biol. 1(6), 362–8.

78. West, M. A. et al. (2004). Science 305(5687), 1153–7.

79. Blander, J. M. et al. (2004). Science 304(5673), 1014–18.

80. Shiratsuchi, A. et al. (2004). J. Immunol. 172(4), 2039–47.

81. Gagnon, E. et al. (2002). Cell 110(1), 119–31.

82. Garin, J. et al. (2001). J. Cell Biol. 152, 165–80.

83. Guermonprez, P. et al. (2003). Nature 425(6956), 397–402.

84. Ackerman, A. L. et al. (2003). Proc. Natl Acad. Sci. USA 100(22), 12889–94.

85. Tsai, B. et al. (2002). Nat. Rev. Mol. Cell Biol. 3(4), 246–55.

86. Tortorella, D. et al. (2000). Annu. Rev. Immunol. 18, 861–926.

87. Wiertz, E. J. et al. (1996). Nature 384(6608), 432–8.

88. Tirosh, B. et al. (2003). J. Biol. Chem. 278(9), 6664–72.

89. Lilley, B. N. et al. (2004). Nature 429(6994), 834–40.

90. Ye, Y. et al. (2004). Nature 429(6994), 841–7.

91. Ye, Y. et al. (2001). Nature 414(6864), 652–6.

92. Houde, M. et al. (2003). Nature 425(6956), 402–6.

93. Ackerman, A. L. et al. (2005). Nat. Immunol. 6(1), 107–13.

94. Roy, C. R. (2005). Proc. Natl Acad. Sci. U S A 102(5), 1271–2.

95. Celli, J. et al. (2003). J. Exp. Med. 198(4), 545–56.

96. Celli, J. et al. (2004). Curr. Opin. Microbiol. 7(1), 93–7.

97. Nagai, H. et al. (2002). Science 295(5555), 679–82.

98. Kagan, J. C. et al. (2004). J. Exp. Med. 199(9), 1201–11.

99. McNew, J. A. et al. (2000). Nature 407(6801), 153–9.

100. Becker, T. et al. (2005). Proc. Natl Acad. Sci. U S A 102(11), 4022–6.

101. Trombetta, E. S. et al. (2005). Annu. Rev. Immunol. 23, 975–1028.

102. Wilson, N. S. et al. (2005). Adv. Immunol. 86, 241–305.

103. Lutz, M. B. et al. (1997). J. Immunol. 159(8), 3707–16.

104. Pierre, P. et al. (1998). Cell 93(7), 1135–45.

105. Trombetta, E. S. et al. (2003). Science 299(5611), 1400–3.

106. Wilson, N. S. et al. (2004). Blood 103(6), 2187–95.

107. Delamarre, L. et al. (2005). Science 307(5715), 1630–4.

�75

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

108. Ramachandra, L. et al. (1999). J. Immunol. 162(6), 3263–72.

109. Ramachandra, L. et al. (2000). J. Immunol. 164(10), 5103–12.

110. Ramachandra, L. et al. (2001). J. Exp. Med. 194(10), 1421–32.

111. Geginat, G. et al. (2001). J. Immunol. 166(3), 1877–84.

112. Skoberne, M. et al. (2002). J. Immunol. 168(4), 1854–60.

113. Skoberne, M. et al. (2002). J. Immunol. 169(3), 1410–18.

114. Tewari, M. K. et al. (2005). Nat. Immunol. 6(3), 287–94.

115. Dani, A. et al. (2004). J. Cell Sci. 117(Pt 18), 4219–30.

116. Cuervo, A. M. et al. (1998). J. Mol. Med. 76(1), 6–12.

117. Zhou, D. et al. (2005). Immunity 22(5), 571–81.

118. Paludan, C. et al. (2005). Science 307(5709), 593–6.

119. Dengjel, J. et al. (2005). Proc. Natl Acad. Sci. U S A 102(22), 7922–7.

120. Yoshimori, T. (2004). Biochem. Biophys. Res. Commun. 313(2), 453–8.

121. Macpherson, A. J. et al. (2004). Science 303(5664), 1662–5.

122. Macpherson, A. J. et al. (2000). Science 288(5474), 2222–6.

123. Garcia-Del Portillo, F. et al. (2000). Infect. Immun. 68(5), 2985–91.

124. Scott, C. C. et al. (2003). J. Membr. Biol. 193(3), 137–52.

125. Sturgill-Koszycki, S. et al. (1994). Science 263(5147), 678–81.

126. Vergne, I. et al. (2004). Annu. Rev. Cell Dev. Biol. 20, 367–94.

127. Fratti, R. A. et al. (2001). J. Cell Biol. 154(3), 631–44.

128. Fratti, R. A. et al. (2003). Proc. Natl Acad. Sci. U S A 100(9), 5437–42.

129. Vergne, I. et al. (2003). J. Exp. Med. 198(4), 653–9.

130. Vergne, I. et al. (2005). Proc. Natl Acad. Sci. U S A 102(11), 4033–8.

131. Gordon, A. H. et al. (1994). Infect. Immun. 62(10), 4650–1.

132. Harding, C. V. et al. (2003). Curr. Opin. Immunol. 15(1), 112–19.

133. Ramachandra, L. et al. (2005). Infect. Immun. 73(2), 1097–105.

134. Hmama, Z. et al. (1998). J. Immunol. 161(9), 4882–93.

135. Chan, J. et al. (2004). Clin. Immunol. 110(1), 2–12.

136. Jantsch, J. et al. (2003). Cell Microbiol. 5(12), 933–45.

137. Cheminay, C. et al. (2005). J. Immunol. 174(5), 2892–9.

138. Tobar, J. A. et al. (2004). J. Immunol. 173(6), 4058–65.

139. Tailleux, L. et al. (2003). J. Immunol. 170(4), 1939–48.

140. Neild, A. L. et al. (2003). Immunity 18(6), 813–23.

141. Bevan, M. J. (1976). J. Exp. Med. 143(5), 1283–8.

142. Heath, W. R. et al. (2004). Immunol. Rev. 199, 9–26.

143. Yewdell, J. W. et al. (2005). Annu. Rev. Immunol. 23, 651–82.

144. Ramirez, M. C. et al. (2002). J. Immunol. 169(12), 6733–42.

145. Fonteneau, J. F. et al. (2003). Blood 102(13), 4448–55.

146. Guermonprez, P. et al. (2005). Springer Semin. Immunopathol. 26(3),

257–71.

�

76

L.

BO

UG

NE

RE

S-V

ER

MO

NT

AN

DP.

GU

ER

MO

NP

RE

Z

147. Shen, L. et al. (2004). Proc. Natl Acad. Sci. U S A 101(9), 3035–40.

148. Wolkers, M. C. et al. (2004). Science 304(5675), 1314–17.

149. Norbury, C. C. et al. (2004). Science 304(5675), 1318–21.

150. Blachere, N. E. et al. (2005). PLoS Biol. 3(6), e185.

151. Binder, R. J. et al. (2005). Nat. Immunol. 6(6), 593–9.

152. Srivastava, P. (2002). Nat. Rev. Immunol. 2(3), 185–94.

153. Tobian, A. A. et al. (2004). J. Immunol. 172(9), 5277–86.

154. Schaible, U. E. et al. (2003). Nat. Med. 9(8), 1039–46.

155. Wijburg, O. L. et al. (2002). J. Immunol. 169(6), 3275–83.

156. Wong, P. et al. (2003). Immunity 18(4), 499–511.

157. Janda, J. et al. (2004). J. Immunol. 173(9), 5644–51.

�77

MH

CC

LA

SS

IA

ND

IIP

AT

HW

AY

SF

OR

PR

ES

EN

TA

TIO

NA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

PART II Dendritic cells and innate immuneresponses to bacteria

CHAPTER 4

Dendritic cell activation and uptakeof bacteria in vivo

Maria RescignoEuropean Institute of Oncology

4.1 INTRODUCTION

Pathogenic bacteria have evolved several strategies to gain access

across epithelial surfaces particularly those lining the mucosae. After

their epithelial transcytosis bacteria find a first line of immune defense

represented by professional phagocytes, including macrophages and dendri-

tic cells. These cells are particularly apt at bacterial uptake, killing and

processing for the initiation/maintenance of adaptive immune responses.

Furthermore, intracellular bacteria can induce by epithelial cells the release

of inflammatory mediators and cytokines that will recruit other immune

cells, particularly neutrophils. Dendritic cells are not simply passive players

waiting for possible invaders, they can actively participate to bacterial

sampling by intercalating between epithelial cells. This mechanism is not

restricted to pathogenic bacteria. Since gut dendritic cells have been

thoroughly studied, in this chapter we will focus on dendritic cells located

in the intestinal mucosa and on their role in the uptake and handling of

luminal bacteria.

4.2 THE ANATOMY OF THE INTESTINAL MUCOSAL EPITHELIUM

AND THE GUT ASSOCIATED LYMPHOID TISSUE (GALT)

The intestinal epithelium is the first line of defense toward dangerous

microorganisms1,2. It opposes a physical, electric and chemical barrier

against luminal bacteria. The permeability of the barrier is regulated by the

presence of both tight junctions (TJ) between epithelial cells (ECs) and

a negatively charged mucous glycocalix. TJ seal adjacent ECs to one another

and regulate solute and ion flux between cells3. The glycocalix sets the size

of macromolecules that can reach the apical membrane of ECs 4 and opposes

an electric barrier to bacteria. Finally, ECs and Paneth cells, specialized cells

located at the base of the crypt of intestinal villi, release antimicrobial

peptides including defensins and cathelicidins that target broad classes

of microorganisms5. The intestinal epithelial barrier is further complicated

by the presence of two important cell types that are interspersed between

ECs and play a crucial role in sampling the luminal content: (microfold)

M cells6 and DCs7�9. M cells are found primarily in the follicle-associated

epithelium (FAE) of Peyer’s patches (PP) but they have been recently

described to be scattered also among the absorptive epithelium where they

could potentially transport antigens to the lamina propria (LP)10. M cells,

differently from ECs, do not have an organized brush border and are more

permissive to antigen uptake4. DCs are phagocytic cells that are scattered

throughout the intestinal epithelium11. It has been recently described that

DCs are able to send dendrites out like periscopes into the lumen for bacterial

uptake12,13. The integrity of the epithelial barrier is preserved because

DCs express TJ proteins and can establish new TJ-like structure with adjacent

ECs12. These ‘‘creeping’’ DCs are characterized by the expression of the

myeloid marker CD11b and the lack of CD8a13,14. Their presence in the

terminal ileum where the gradient of bacteria gradually increases suggests

they may be recruited by the presence of luminal bacteria. Interestingly,

DCs in CX3CL1 (fractalkine) receptor-deficient mice are unable to spread

their dendrites across the epithelial barrier, indicating the involvement

of CX3CL1 in driving the extension of the dendrites14. It is unknown whether

bacteria can directly drive fractalkine production by epithelial cells and

whether fractalkine modulates TJ protein expression in DCs. Interestingly,

bacteria lacking LPS are unable to recruit DCs in in vitro generated epithelial

cell monolayers suggesting that bacteria play an active role in the induction

of DC migration across the epithelial barrier15.

The GALT can be divided into inductive sites where the immune

response is initiated and effector sites where immune cells carry out their

function2,16. Peyer’s patches, mesenteric lymph nodes (MLN) and isolated

lymphoid follicles are important inductive sites for mucosal immune

responses whereas the epithelium and the lamina propria of the mucosa

are considered effector sites for antibody production and T cell responses.

4.3 BACTERIAL UPTAKE IN THE GUT AND MUCOSAL DC

SUBPOPULATIONS

In the absence of concomitant activation stimuli, LP-DCs are probably

involved in the induction of oral tolerance. In fact expansion of DCs in vivo

�

82

M.

RE

SC

IGN

O

enhanced tolerance induction after antigen feeding17. It is possible that

antigen-loaded DCs migrate to MLN which is the preferential site for naive

T cell activation and expansion after oral feeding of soluble antigen18.

Conversely, particulate antigen is most likely taken up in PP as mice lacking

PP are perfectly competent to induce antibody response toward soluble

but not toward particulate (microsphere) antigen19.

The mechanisms of bacterial entrance depend on their pathogenicity

(Figure 4.1). Most of the pathogens have developed strategies to penetrate

ECs or to facilitate M cell invasion (for a review see Ref. 1), whereas

non-invasive bacteria can enter mucosal surfaces either through M cells

or DCs. M cells can release their ‘‘cargo’’ to underlying phagocytic cells,

including DCs, that can migrate to the interfollicular region of PP for T

and B cell interactions, whereas DCs that take up bacteria directly across

mucosal surfaces are likely to migrate to MLN. Interestingly, MLN set the

border for mucosal compartment avoiding systemic spread of commensal-

loaded DCs20. Both mechanisms do not discriminate between invasive

pathogenic and non-invasive commensal bacteria. An alternative mechan-

ism for antigen entry across a mucosal surface that also targets DCs

and could be used for bacterial internalization, has been recently described21.

It is mediated by neonatal Fc receptors (FcRn) expressed by adult human

(but not mouse) intestinal epithelial cells that transport IgG across the

intestinal epithelial barrier, and after binding with cognate antigen in the

intestinal lumen, recycles the immune complexes back to the LP21. Antigens

bound by IgG are less susceptible to degradation within the epithelial

cells because endosomes formed after uptake by FcRn do not readily fuse

with lysosomes. FcRn transport directs and delivers the antigens in the

form of immune complexes directly to DCs lying in the LP. As DCs can

be activated by immune complexes, it would be interesting to know

whether DCs internal-ize the immune complexes via the FcgRs or via

FcRn (both of which are expressed by DCs) and whether these receptors

differentially affect DC function. The role of IgA and their secretory

form (sIgA) in facilitating the internalization of opsonized bacteria still

needs to be investigated but it is known that IgA-coated antigens although

being excluded from epithelial cell binding, are facilitated in their access

across M cells22 and it has been recently shown that they are targeted

directly to DCs present in the dome region of PPs23. Finally, DCs can

process antigens from apoptotic intestinal epithelial cells, both in the

steady state24 and following reovirus infection25, which constitutes another

mechanism of DC antigen uptake that directly involves interactions with

the epithelium.

�83

DE

ND

RIT

ICC

EL

LA

CT

IVA

TIO

NA

ND

UP

TA

KE

OF

BA

CT

ER

IAIN

VIV

O

The uptake route together with the nature of the ingested antigens

dictates the type of immune response that is generated, whether this

is related to the subtype of DCs that is targeted by each route or to their

location remains to be established. In fact at least four DC populations in

the mouse intestine have been described. They are all characterized by

the expression of CD11c but differ for the expression of the surface

markers CD11b, CD8a and B220 (for a review see Refs 8 and 26) as well

as for the expression of chemokine receptors CCR6 and CCR727.

Figure 4.1. Mechanisms of bacterial uptake. The mechanisms of bacterial entrance depend

on their pathogenicity. Most of the pathogens have developed strategies to penetrate ECs or to

facilitate M cell invasion via the expression of type three secretion system and invasive genes;

alternatively they are captured by creeping DCs (left). Commensal bacteria can enter mucosal

surfaces either through M cells or DCs (right). It is not yet clear whether entrance through M

cells of commensal bacteria is restricted to immunoglobulin-coated bacteria. In this case,

internalization of IgG-coated bacteria could be mediated by neonatal Fc receptors (FcRn)

expressed by adult human (but not mouse) intestinal epithelial cells, whereas secretory

(s)IgA-coated bacteria could be internalized by IgA receptor expressed by M cells. M cells can

release their ‘‘cargo’’ to underlying phagocytic cells, including DCs, that can migrate to the

interfollicular region (IFR) of Peyer’s Patches for Tand B cell interactions, whereas DCs that

take up bacteria directly across mucosal surfaces are likely to migrate to MLN. Alternatively,

PP-DCs could migrate to MLN. HEV: high endothelial venules. (For a colour version of this

figure, please refer to the colour insert between pages 12 and 13.)�

84

M.

RE

SC

IGN

O

Interestingly, the different DC populations have particular locations in PP28.

In fact, it is important to say that in PP two important functions are carried

out by DCs: uptake of antigen after its transcytosis across the FAE and T and

B cell activation. Therefore, differently from other peripheral tissues, it is

possible to find in the PP both immature DCs that are mainly localized in the

sub-epithelial dome, below the FAE and mature DCs that are found in

interfollicular T cell areas. Two additional DC subsets have been described in

MLN that are characterized by the differential expression of CD4 and

DEC-2058,26. The characterization of human intestinal DCs is still very poor,

but at least two DC types have been described in the colon: a CD11cþHLA-

DRþ population and a CD11c� population29 that we have identified as

CD83þCD123þ, possibly plasmacytoid DCs (our unpublished observations).

Hence, scattered throughout mucosal tissues it is possible to find the same

DC subsets present in other non-mucosal tissues (see Chapter 1).

4.4 HANDLING OF BACTERIA BY EPITHELIAL CELLS CAN

INFLUENCE THE INDUCTION OF IMMUNE RESPONSES

The major interaction between mucosal tissues and luminal bacteria

occurs at the level of ECs that are the most representative cell type of the

epithelium. Both pathogens and commensal bacteria have been described

to undertake an active cross-talk with ECs1. Whereas the first are primarily

involved in the activation of an inflammatory cascade of events, the latter

seem to downregulate the ability of ECs to initiate inflammatory responses.

The mechanisms through which pathogens can activate ECs are similar to

those used by monocytes and DCs to sense the presence of bacteria. In fact

ECs express a series of pathogen recognition receptors (PRRs, see Chapters 2

and 9) including Toll-like receptors (TLRs) and NOD proteins that are

expressed also by phagocytes1. The major difference stands in the location

of these receptors. In fact ECs seem to express these receptors either

intracellularly (like TLR-4) or in a polarized fashion leaving the apical surface

nearly free of PRR expression. Therefore only invasive bacteria or those

equipped with type three or four secretion systems30 that act as syringes

to pump DNA or effector proteins directly into the cytoplasm of host cells,

are sensed by PRRs for activation of the inflammatory cascade. Moreover,

some of the receptors (like NOD2) are constitutively expressed only in Paneth

cells31 that reside at the base of the cripts and are induced in ECs only after

bacterial encounter32,33. A typical indicator of epithelial infection by invasive

bacteria is the expression of the chemokine CXCL-8 (IL-8) which is a strong

�85

DE

ND

RIT

ICC

EL

LA

CT

IVA

TIO

NA

ND

UP

TA

KE

OF

BA

CT

ER

IAIN

VIV

O

chemoattractant for neutrophils34�37. A more debated issue relates to the

expression of TLR-5, the receptor for flagellin38. It has been recently

described that flagellin-dependent stimulation of intestinal ECs results

in triggering of CCL20 via a TLR-5 dependent mechanism39. CCL20 is

responsible for the recruitment of CCR6-expressing immature DCs40.

However, some authors suggest that TLR-5 is expressed only basolaterally

of ECs41,42, whereas others have also described it apically39,43. We favor the

second hypothesis because we have evidence that invasive-deficient mutant

of Salmonella and the flagellated non-invasive soil bacterium Bacillus subtilis

induce the expression of CCL-20 by polarized ECs15,44. Our experiments in

the mouse also confirm that non-invasive flagellated bacteria can induce the

expression of CCL-20 suggesting the possibility that different responses

might depend on the EC cell line used for in vitro experiments44.

How commensals can downregulate the inflammatory response

induced by pathogen associated molecular patterns (PAMPs) has only

recently started to be unraveled. It is becoming clear that recognition

of commensal flora via TLRs is required for intestinal homeostasis45 and

that commensal bacteria can interfere at different levels of TLR signaling.

Expression and activation of IRAK-M46 or of a truncated version of the

TLR adaptor protein MyD8847 that both interfere with TLR signaling have

been described. Along the same line, the interaction of ECs with the

commensal Bacteroides thetaiotamicron or with non-virulent mutants of

Salmonella typhimurium interfere with the activation of NF-kB that is

downstream of TLR signaling either by triggering binding of peroxisome-

proliferator-activated receptor g (PPAR-g) with the NF-kB subunit Rel-A in

the nucleus48 or by blocking the degradation of IkBa, an intracellular

inhibitor of NF-kB49. Therefore, the induction of an inflammatory response

in ECs depends on the ability of invasive pathogens to activate PRR signaling

pathways and on that of commensals to perturb the same signaling

pathways.

4.5 UNIQUE FUNCTIONS OF MUCOSAL DCS

DCs isolated from a variety of mucosal sites (PP, LP, mesenteric lymph

nodes (MLN), lung) have the natural propensity to induce TH2 responses in

in vitro T cell priming assays, and to express cytokines such as IL-10, and

possibly TGF-b2,50�53. Interestingly, the same CD11cþCD11bþCD8a� DC

subset isolated from PP but not from spleen preferentially polarizes

�

86

M.

RE

SC

IGN

O

antigen-specific T cells to produce TH2 cytokines and IL-10 in vitro54,

suggesting that the observed differences are not attributable to subset-

intrinsic properties but most likely to the local mucosal microenvironment.

Further, the same PP but not spleen DC subset is able to promote IgA

production by naive B cells, which is mediated by a higher release of IL-655

and T cell help. These data suggest that mucosal DCs may be specialized in

inducing a non-inflammatory environment and in providing help to B cells

via the activation of TH2 T cells. This is consistent with the fact that many

‘‘tolerogenic’’ responses to mucosal antigens, for example to commensal

organisms, are associated with the generation of antibody responses20,51,

rather than with a broad immunological unresponsiveness. In addition,

CD8þ CD11clo plasmacytoid DCs may also be important for maintaining

tolerance to innocuous antigens since this population can induce the

differentiation of IL-10 producing regulatory T cells (Treg) in vitro8.

Another important feature of DCs isolated from mucosal tissues is that

they have the unique ability to selectively imprint gut-homing T cells56�58.

Moreover, naive CD8þ T cells primed by PP-DCs acquire gut tropism57,

despite showing similar patterns of activation markers and effector activity as

those primed by DCs isolated from other non-mucosal lymphoid organs.

PP-DCs induced high expression of the intestinal homing integrin a4b7 and

the chemokine receptor CCR9 in primed CD8þ T cells. Interestingly,

reactivation of skin-committed memory T cells with DCs isolated from gut

changed T cell tissue tropism, suggesting that memory T cells are relocated

according to the tissue where they are needed59. Finally, mucosal DCs have

been shown to continually migrate to draining lymph nodes in the ‘‘steady’’,

or unperturbed-state with a rapid turnover rate (2�4 days in the intestinal

wall). In the rat, two types of migrating DCs could be identified, both

of which are positive for the aE integrin CD103, but only the fraction

that expresses low levels of CD172 (SIRPa), has features of immature cells

and carries apoptotic enterocytes to MLNs60. Because these DCs process

apoptotic epithelial cells in the steady-state24, this CD103þCD172lo

DC population may be involved in tolerance to self-proteins, although this

hypothesis remains to be tested. DC emigration from the gut can be

greatly enhanced by systemic LPS injection which does not change the

proportion of SIRPahi/SIRPalo populations as well as their activation state61.

Interestingly, whereas SIRPalo DCs migrate to T cell areas of MLNs

under steady-state conditions, SIRPahi DCs do so only after intravenous

LPS injection suggesting that LPS injection facilitates antigen presentation

by this DC subset.

�87

DE

ND

RIT

ICC

EL

LA

CT

IVA

TIO

NA

ND

UP

TA

KE

OF

BA

CT

ER

IAIN

VIV

O

4.6 THE CROSS-TALK BETWEEN ECS AND DCS HELPS

REGULATING THE INDUCTION OF MUCOSAL IMMUNE

RESPONSES

DCs play an active role in bacterial uptake across mucosal surfaces and

have unique functions that allow the generation of mucosal immune

responses. Moreover, DCs can intercalate between ECs and can interact

directly with the luminal bacteria and with all the TLR ligands that are carried

by commensal or pathogenic bacteria. Therefore, three important questions

arise: what is the role played by the local microenvironment in driving

mucosal DC differentiation? How can DCs avoid the induction of exag-

gerated inflammatory responses toward commensal bacteria? Is there

any relationship between the unique phenotype of mucosal DCs and the

regulation of gut immune homeostasis? One possibility is that DCs sense

differently commensal versus pathogenic bacteria. However, rather than

a difference between dangerous and non-dangerous bacteria it seems that

Gram negative versus Gram positive bacteria are differentially sensed by

DCs. In fact, Gram negative bacteria (including commensals) activate DCs

that induce a Th1 type of response, by contrast, Gram positive bacteria are

more prone to induce Th2 type of responses62. Certain probiotics belonging

to the Lactobacillus species (L. reuteri and L. casei, but not L. plantarum) can

drive tolerogenic DCs63. Therefore, it is not possible to generalize on the

possibility of DCs to sense differently commensals, but some species could

participate in downmodulating DC function. It is becoming clear that the

relationship between DCs and the microenvironment are profoundly

affecting the functional properties of tissue DCs. This has been demon-

strated in the spleen64,65, but there is strong evidence that a similar situation

is occurring also in the gut. In fact, the ability of intestinal DCs to induce gut-

tropism during T cell priming56�58 and reactivation59 and to promote TH2

T cell responses2,50�54, as well as IgA antibody production55 strongly favors

this hypothesis. As intestinal ECs are in close contact with DCs, they could

play an active role in driving mucosal DC differentiation. This is indeed the

case because ECs release constitutively thymic stromal lymphopoietin

(TSLP), a molecule involved in driving TH2 differentiation by DCs66,67.

Interestingly, DCs exposed to EC-conditioning are unable to release IL-12

and to drive TH1 type of T cell responses even after activation with TH1-

inducing pathogens (Figure 4.2)68. Moreover, TSLP acts in a very narrow

window of concentrations: at lower or higher TSLP concentrations, DCs

reacquire the ability to release IL-12 and to drive TH1 T cell responses.

Therefore, it is likely that resident DCs even though they have the chance to

�

88

M.

RE

SC

IGN

O

Figure 4.2. The cross-talk between ECs and DCs in Salmonella typhimurium handling.

Early Salmonella typhimurium infection: resident DCs are conditioned by EC-released

TSLP (Res-DC). Res-DC release IL-10 after bacterial exposure and drive default TH2

responses to S. typhimurium. It remains to be established whether these cells also drive

T regulatory cells. Late infection: since Salmonella typhimurium is an invasive bacterium, it

induces ECs to release pro-inflammatory chemokines like IL-8 (CXCL-8) and PARC

(CCL-18), which attract neutrophils, granulocytes and activated T cells that generate an

inflamed site. The binding of Salmonella to the basolateral membrane of ECs induces

the upregulation of TSLP. TSLP at this concentration drives TH1 rather than TH2

promoting DCs in response to bacteria. Unidentified EC-derived factors can also activate

‘‘bystander’’ DCs that have not been in contact directly with the bacteria. DCs activated in

this way release IL-10 and TARC (CCL-17) but not IL-12, thus driving and recruiting TH2

Tcells and maybe Tregulatory cells. Salmonella also induces the release of MIP-3a (CCL-20)

that recruits CCR6-expressing immature DCs. Most likely, recruited DCs are not subjected

to EC-conditioning, rather they could find increased TSLP concentrations in the infected

site. Newly recruited DCs (Rec-DC) can either creep between ECs to take up bacteria or

they can phagocytose bacteria that have breached across the epithelial barrier and release

both IL-10 and IL-12, thus promoting TH1 and TH2 responses. This allows the establish-

ment of protective anti-Salmonella responses. (For a colour version of this figure, please

refer to the colour insert between pages 12 and 13.)

contact directly the bacteria, they are unable to activate inflammatory cells

and this can help to maintain the homeostasis of the gut. In fact, nearly 70

per cent of individuals affected by a TH1-mediated chronic inflammatory

disease like in Crohn’s disease69 have undetectable levels of TSLP and this

correlates with the inability of intestinal ECs to regulate DC function68.

Therefore, resident DCs that are actively involved in taking up bacteria at

steady-state do not drive inflammatory responses and this can explain why

the intestinal immune homeostasis is preserved even though DCs are

continuously exposed to TLR ligands.

Despite this propensity for the induction of TH2 and Tregs by mucosal

DCs, TH1 and cytotoxic T lymphocyte (CTL) responses are effectively

generated to mucosal pathogens and are required to fight intracellular

microorganisms70�74. Whether this involves the same or different DC

subsets as those responsible for mucosal responses and tolerance induction,

remains to be established. However, it is conceivable that resident mucosal

DCs are ‘‘educated’’ by ECs to initiate non-inflammatory responses, whereas

DCs recruited after bacterial invasion might retain their ability to respond in

an inflammatory mode. In fact, infection by flagellated bacteria like

Salmonella spp. induces the recruitment of DCs in the intestinal

epithelium12,14 via the release of CCL-20 by ECs39. These non-conditioned

newly recruited DCs might be responsible for the induction of TH1 responses

to invasive bacteria (Figure 4.2). This hypothesis is supported by in vitro

three-partite studies in which DCs were seeded from the basolateral

membrane of EC monolayers shortly before apical bacterial application68.

Interestingly, due to their ability to creep between ECs and to contact bacteria

directly, DCs were ‘‘qualitatively’’ similarly activated regardless of the

invasiveness or pathogenicity of the apical bacteria. Bacteria-activated DCs

produced both IL-12 and IL-10 and skewed toward a TH1 phenotype68. This

suggests that non-conditioned DCs can drive the induction of inflammatory

responses provided that they are not subject to EC conditioning before

their encounter with bacteria. Moreover, bacteria invading ECs induce the

upregulation of TSLP thus switching to DCs that have the propensity to

induce TH1- rather than TH2-T cells in response to bacteria. Interestingly,

bystander DCs that do not contact directly the bacteria are activated by

EC-derived factors to non-inflammatory DCs producing IL-10 and TARC

(CCL-17) and inducing or recruiting TH2 T cells, probably as a feedback

mechanism to turn off the inflammatory response44. Whether these DCs

also drive T regulatory cells has to be investigated.

Another possibility is that epithelial cell derived factors, such as TNF

or type 1 IFNs, produced during pathogen invasion may directly affect

�

90

M.

RE

SC

IGN

O

DC activation. This hypothesis is supported by studies of murine intestinal

infection with type-1 reovirus25. Reovirus productively infects epithelial cells

overlying PPs, yet viral antigen associated with apoptotic epithelial cells is

avidly taken up by CD11cþ CD8a� CD11b� DCs in the subepithelial dome25.

The observation that reovirus neither productively infects DCs in vivo or in

vitro, nor activates DCs to mature or produce cytokines in vitro, suggests a

role for environmental factors, possibly derived from infected epithelial cells,

in driving DCs to induce TH1 responses to the virus. Interestingly, IFNabR-

deficient mice, but not MyD88-deficient or TLR3-deficient mice have an

increased susceptibility to reovirus infection. In addition, MyD88-deficient

mice mount normal IgG1, IgG2a/c and IgG2b responses, suggesting that

type 1 IFN, possibly derived in the early stages of infection from infected

epithelial cells, but not signaling via at least a single TLR pathway is

important for inducing protection from reovirus infection.

Taken together, these studies highlight an important emerging relation-

ship between DCs and epithelial cells in the maintenance of mucosal

homeostasis and the induction of innate and adaptive immunity to mucosal

infection with pathogens.

4.7 CONCLUSIONS

In conclusion, the outcome of bacterial handling by DCs at mucosal surfaces

depends on several factors. These include: (a) the ability of DCs to discrimi-

nate between different sorts of bacteria through differential TLR engage-

ment; (b) the unique specialized functions of mucosal DCs that allow the

establishment of mucosal immune responses, including the induction of

TH2 T cell responses and IgA antibody production; (c) the interplay between

epithelial cells and DCs at steady-state and during infection. Therefore,

DCs play a crucial role both in the uptake of intestinal bacteria and in the

induction of tolerance and immunity towards them. However, it remains

to be clarified whether different DC subsets have clearly distinct functions

in vivo or whether the local microenvironment is responsible to control

DC function.

REFERENCES

1. Sansonetti, P. J. (2004). War and peace at mucosal surfaces. Nat. Rev.

Immunol. 4, 953�64.

2. Mowat, A. M. (2003). Anatomical basis of tolerance and immunity to

intestinal antigens. Nat. Rev. Immunol. 3, 331�41.

�91

DE

ND

RIT

ICC

EL

LA

CT

IVA

TIO

NA

ND

UP

TA

KE

OF

BA

CT

ER

IAIN

VIV

O

3. Schneeberger, E. E. and Lynch, R. D. (2004). The tight junction: a

multifunctional complex. Am. J. Physiol. Cell Physiol. 286, C1213�28.

4. Frey, A., Giannasca, K. T., Weltzin, R., Giannasca, P. J., Reggio, H., Lencer,

W. I. and Neutra, M. R. (1996). Role of the glycocalyx in regulating access of

microparticles to apical plasma membranes of intestinal epithelial cells:

implications for microbial attachment and oral vaccine targeting.

J. Exp. Med. 184, 1045�59.

5. Ganz, T. (2003). Defensins: antimicrobial peptides of innate immunity.

Nat. Rev. Immunol. 3, 710�20.

6. Kraehenbuhl, J. P. and Neutra, M. R. (2000). Epithelial M cells:

differentiation and function. Annu. Rev. Cell Dev. Biol. 16, 301�32.

7. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J.,

Pulendran, B. and Palucka, K. (2000). Immunobiology of dendritic cells.

Annu. Rev. Immunol. 18, 767�811.

8. Bilsborough, J. and Viney, J. L. (2004). Gastrointestinal dendritic cells

play a role in immunity, tolerance, and disease. Gastroenterology 127,

300�9.

9. Kelsall, B. L. and Rescigno, M. (2004). Mucosal dendritic cells in

immunity and inflammation. Nat. Immunol. 5, 1091�5.

10. Jang, M. H., Kweon, M. N., Iwatani, K., Yamamoto, M., Terahara, K.,

Sasakawa, C., Suzuki, T., Nochi, T., Yokota, Y., Rennert, P. D. et al. (2004).

Intestinal villous M cells: an antigen entry site in the mucosal epithelium.

Proc. Natl Acad. Sci. U S A 101, 6110�15.

11. Maric, I., Holt, P. G., Perdue, M. H. and Bienenstock, J. (1996). Class II

MHC antigen (Ia)-bearing dendritic cells in the epithelium of the rat

intestine. J. Immunol. 156, 1408�14.

12. Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G.,

Bonasio, R., Granucci, F., Kraehenbuhl, J. P. and Ricciardi-Castagnoli, P.

(2001). Dendritic cells express tight junction proteins and penetrate gut

epithelial monolayers to sample bacteria. Nat. Immunol. 2, 361�7.

13. Rescigno, M., Rotta, G., Valzasina, B. and Ricciardi-Castagnoli, P. (2001).

Dendritic cells shuttle microbes across gut epithelial monolayers.

Immunobiology 204, 572�81.

14. Niess, J. H., Brand, S., Gu, X., Landsman, L., Jung, S., McCormick, B. A.,

Vyas, J. M., Boes, M., Ploegh, H. L., Fox, J. G. et al. (2005). CX3CR1-

mediated dendritic cell access to the intestinal lumen and bacterial

clearance. Science 307, 254�8.

15. Rimoldi, M., Chieppa, M., Vulcano, M., Allavena, P. and Rescigno, M.

(2004). Intestinal epithelial cells control DC function. Ann. N Y Acad. Sci.

1029, 1�9.

�

92

M.

RE

SC

IGN

O

16. Nagler-Anderson, C. (2001). Man the barrier! Strategic defences in the

intestinal mucosa. Nat. Rev. Immunol. 1, 59�67.

17. Viney, J. L., Mowat, A. M., O’Malley, J. M., Williamson, E. and Fanger, N. A.

(1998). Expanding dendritic cells in vivo enhances the induction of

oral tolerance. J. Immunol. 160, 5815�25.

18. Kunkel, D., Kirchhoff, D., Nishikawa, S., Radbruch, A. and Scheffold, A.

(2003). Visualization of peptide presentation following oral application of

antigen in normal and Peyer’s patches-deficient mice. Eur. J. Immunol. 33,

1292�301.

19. Kunisawa, J., Takahashi, I., Okudaira, A., Hiroi, T., Katayama, K.,

Ariyama, T., Tsutsumi, Y., Nakagawa, S., Kiyono, H. and Mayumi, T.

(2002). Lack of antigen-specific immune responses in anti-IL-7 receptor

alpha chain antibody-treated Peyer’s patch-null mice following intestinal

immunization with microencapsulated antigen. Eur. J. Immunol. 32,

2347�55.

20. Macpherson, A. J. and Uhr, T. (2004). Induction of protective IgA by

intestinal dendritic cells carrying commensal bacteria. Science 303, 1662�5.

21. Yoshida, M., Claypool, S. M., Wagner, J. S., Mizoguchi, E., Mizoguchi, A.,

Roopenian, D. C., Lencer, W. I. and Blumberg, R. S. (2004). Human

neonatal Fc receptor mediates transport of IgG into luminal

secretions for delivery of antigens to mucosal dendritic cells. Immunity 20,

769�83.

22. Weltzin, R., Lucia-Jandris, P., Michetti, P., Fields, B. N., Kraehenbuhl, J. P.,

and Neutra, M. R. (1989). Binding and transepithelial transport of

immunoglobulins by intestinal M cells: demonstration using

monoclonal IgA antibodies against enteric viral proteins. J. Cell Biol. 108,

1673�85.

23. Rey, J., Garin, N., Spertini, F. and Corthesy, B. (2004). Targeting of secretory

IgA to Peyer’s patch dendritic and T cells after transport by intestinal M

cells. J. Immunol. 172, 3026�33.

24. Huang, F. P., Platt, N., Wykes, M., Major, J. R., Powell, T. J., Jenkins, C. D.

and MacPherson, G. G. (2000). A discrete subpopulation of dendritic cells

transports apoptotic intestinal epithelial cells to T cell areas of mesenteric

lymph nodes, see comments. J. Exp. Med. 191, 435�44.

25. Fleeton, M. N., Contractor, N., Leon, F., Wetzel, J. D., Dermody, T. S. and

Kelsall, B. L. (2004). Peyer’s patch dendritic cells process viral antigen from

apoptotic epithelial cells in the intestine of reovirus-infected mice. J. Exp.

Med. 200, 235�45.

26. Shortman, K. and Liu, Y. J. (2002). Mouse and human dendritic cell

subtypes. Nat. Rev. Immunol. 2, 151�61.

�93

DE

ND

RIT

ICC

EL

LA

CT

IVA

TIO

NA

ND

UP

TA

KE

OF

BA

CT

ER

IAIN

VIV

O

27. Iwasaki, A. and Kelsall, B. L. (2000). Localization of distinct Peyer’s patch

dendritic cell subsets and their recruitment by chemokines macrophage

inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid

organ chemokine. J. Exp. Med. 191, 1381�94.

28. Niedergang, F., Didierlaurent, A., Kraehenbuhl, J. P. and Sirard, J. C.

(2004). Dendritic cells: the host Achille’s heel for mucosal pathogens?

Trends Microbiol. 12, 79�88.

29. Bell, S. J., Rigby, R., English, N., Mann, S. D., Knight, S. C., Kamm, M. A.

and Stagg, A. J. (2001). Migration and maturation of human colonic

dendritic cells. J. Immunol. 166, 4958�67.

30. Viala, J., Chaput, C., Boneca, I. G., Cardona, A., Girardin, S. E., Moran,

A. P., Athman, R., Memet, S., Huerre, M. R., Coyle, A. J. et al. (2004). Nod1

responds to peptidoglycan delivered by the Helicobacter pylori cag

pathogenicity island. Nat. Immunol. 5, 1166�74.

31. Lala, S., Ogura, Y., Osborne, C., Hor, S. Y., Bromfield, A., Davies, S.,

Ogunbiyi, O., Nunez, G. and Keshav, S. (2003). Crohn’s disease and the

NOD2 gene: a role for paneth cells. Gastroenterology 125, 47�57.

32. Rosenstiel, P., Fantini, M., Brautigam, K., Kuhbacher, T., Waetzig, G. H.,

Seegert, D. and Schreiber, S. (2003). TNF-alpha and IFN-gamma regulate

the expression of the NOD2 (CARD15) gene in human intestinal epithelial

cells. Gastroenterology 124, 1001�9.

33. Gutierrez, O., Pipaon, C., Inohara, N., Fontalba, A., Ogura, Y., Prosper, F.,

Nunez, G. and Fernandez-Luna, J. L. (2002). Induction of Nod2 in

myelomonocytic and intestinal epithelial cells via nuclear factor-kappa B

activation. J. Biol. Chem. 277, 41701�5.

34. Eckmann, L., Kagnoff, M. F. and Fierer, J. (1993). Epithelial cells secrete the

chemokine interleukin-8 in response to bacterial entry. Infect. Immun. 61,

4569�74.

35. Jung, H. C., Eckmann, L., Yang, S. K., Panja, A., Fierer, J., Morzycka-

Wroblewska, E. and Kagnoff, M. F. (1995). A distinct array of proinflam-

matory cytokines is expressed in human colon epithelial cells in response to

bacterial invasion. J. Clin. Invest. 95, 55�65.

36. McCormick, B. A., Colgan, S. P., Delp-Archer, C., Miller, S. I. and Madara,

J. L. (1993). Salmonella typhimurium attachment to human intestinal

epithelial monolayers: transcellular signalling to subepithelial neutrophils.

J. Cell Biol. 123, 895�907.

37. McCormick, B. A., Hofman, P. M., Kim, J., Carnes, D. K., Miller, S. I. and

Madara, J. L. (1995). Surface attachment of Salmonella typhimurium to

intestinal epithelia imprints the subepithelial matrix with gradients

chemotactic for neutrophils. J. Cell Biol. 131, 1599�608.

�

94

M.

RE

SC

IGN

O

38. Hayashi, F., Smith, K. D., Ozinsky, A., Hawn, T. R., Yi, E. C., Goodlett,

D. R., Eng, J. K., Akira, S., Underhill, D. M. and Aderem, A. (2001). The

innate immune response to bacterial flagellin is mediated by Toll-like

receptor 5. Nature 410, 1099�103.

39. Sierro, F., Dubois, B., Coste, A., Kaiserlian, D., Kraehenbuhl, J. P. and

Sirard, J. C. (2001). Flagellin stimulation of intestinal epithelial cells

triggers CCL20-mediated migration of dendritic cells. Proc. Natl. Acad. Sci.

U S A 98, 13722�7.

40. Sozzani, S., Allavena, P., D’Amico, G., Luini, W., Bianchi, G., Kataura, M.,

Imai, T., Yoshie, O., Bonecchi, R. and Mantovani, A. (1998). Differential

regulation of chemokine receptors during dendritic cell maturation: a

model for their trafficking properties. J. Immunol. 161, 1083�6.

41. Gewirtz, A. T., Simon, P. O., Jr., Schmitt, C. K., Taylor, L. J., Hagedorn,

C. H., O’Brien, A. D., Neish, A. S. and Madara, J. L. (2001). Salmonella

typhimurium translocates flagellin across intestinal epithelia, inducing a

proinflammatory response. J. Clin. Invest. 107, 99�109.

42. Lyons, S., Wang, L., Casanova, J. E., Sitaraman, S. V., Merlin, D. and

Gewirtz, A. T. (2004). Salmonella typhimurium transcytoses flagellin

via an SPI2-mediated vesicular transport pathway. J. Cell Sci.

117, 5771�80.

43. Ramos, H. C., Rumbo, M. and Sirard, J. C. (2004). Bacterial flagellins:

mediators of pathogenicity and host immune responses in mucosa. Trends

Microbiol. 12, 509�17.

44. Rimoldi, M., Chieppa, M., Larghi, P., Vulcano, M., Allavena, P. and

Rescigno, M. (2005). Monocyte-derived dendritic cells activated by bacteria

or by bacteria-stimulated epithelial cells are functionally different. Blood

106, 2818�26.

45. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. and

Medzhitov, R. (2004). Recognition of commensal microflora by Toll-like

receptors is required for intestinal homeostasis. Cell 118, 229�41.

46. Kobayashi, K., Hernandez, L. D., Galan, J. E., Janeway, C. A., Jr., Medzhitov,

R. and Flavell, R. A. (2002). IRAK-M is a negative regulator of Toll-like

receptor signaling. Cell 110, 191�202.

47. Janssens, S., Burns, K., Tschopp, J. and Beyaert, R. (2002). Regulation of

interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by

alternative splicing of MyD88. Curr. Biol. 12, 467�71.

48. Kelly, D., Campbell, J. I., King, T. P., Grant, G., Jansson, E. A., Coutts, A. G.,

Pettersson, S. and Conway, S. (2004). Commensal anaerobic gut bacteria

attenuate inflammation by regulating nuclear-cytoplasmic shuttling of

PPAR-gamma and RelA. Nat. Immunol. 5, 104�12.

�95

DE

ND

RIT

ICC

EL

LA

CT

IVA

TIO

NA

ND

UP

TA

KE

OF

BA

CT

ER

IAIN

VIV

O

49. Neish, A. S., Gewirtz, A. T., Zeng, H., Young, A. N., Hobert, M. E.,

Karmali, V., Rao, A. S. and Madara, J. L. (2000). Prokaryotic regulation of

epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science

289, 1560�3.

50. Akbari, O., DeKruyff, R. H. and Umetsu, D. T. (2001). Pulmonary dendritic

cells producing IL-10 mediate tolerance induced by respiratory exposure to

antigen. Nat. Immunol. 2, 725�31.

51. Alpan, O., Rudomen, G. and Matzinger, P. (2001). The role of dendritic

cells, B cells, and M cells in gut-oriented immune responses. J. Immunol.

166, 4843�52.

52. Iwasaki, A. and Kelsall, B. L. (1999). Freshly isolated Peyer’s patch,

but not spleen, dendritic cells produce interleukin 10 and induce the

differentiation of T helper type 2 cells. J. Exp. Med. 190, 229�39.

53. Williamson, E., Bilsborough, J. M. and Viney, J. L. (2002). Regulation

of mucosal dendritic cell function by receptor activator of NF-kappa B

(RANK)/RANK ligand interactions: impact on tolerance induction.

J. Immunol. 169, 3606�12.

54. Iwasaki, A. and Kelsall, B. L. (2001). Unique functions of cd11b(þ),

cd8alpha(þ), and double-negative Peyer’s patch dendritic cells. J.

Immunol. 166, 4884�90.

55. Sato, A., Hashiguchi, M., Toda, E., Iwasaki, A., Hachimura, S. and

Kaminogawa, S. (2003). CD11bþ Peyer’s patch dendritic cells secrete

IL-6 and induce IgA secretion from naive B cells. J. Immunol. 171,

3684�90.

56. Stagg, A. J., Kamm, M. A. and Knight, S. C. (2002). Intestinal dendritic cells

increase T cell expression of alpha4beta7 integrin. Eur. J. Immunol. 32,

1445�54.

57. Mora, J. R., Bono, M. R., Manjunath, N., Weninger, W., Cavanagh, L. L.,

Rosemblatt, M. and Von Andrian, U. H. (2003). Selective imprinting

of gut-homing T cells by Peyer’s patch dendritic cells. Nature 424, 88�93.

58. Johansson-Lindbom, B., Svensson, M., Wurbel, M. A., Malissen, B.,

Marquez, G. and Agace, W. (2003). Selective generation of gut tropic T

cells in gut-associated lymphoid tissue (GALT): requirement for GALT

dendritic cells and adjuvant. J. Exp. Med. 198, 963�9.

59. Mora, J. R., Cheng, G., Picarella, D., Briskin, M., Buchanan, N. and

von Andrian, U. H. (2005). Reciprocal and dynamic control of CD8 T cell

homing by dendritic cells from skin- and gut-associated lymphoid tissues.

J. Exp. Med. 201, 303�16.

60. Yrlid, U. and Macpherson, G. (2003). Phenotype and function of rat

dendritic cell subsets. Apmis 111, 756�65.

�

96

M.

RE

SC

IGN

O

61. Turnbull, E. L., Yrlid, U., Jenkins, C. D. and Macpherson, G. G. (2005).

Intestinal dendritic cell subsets: differential effects of systemic TLR4

stimulation on migratory fate and activation in vivo. J. Immunol. 174,

1374�84.

62. Smits, H. H., van Beelen, A. J., Hessle, C., Westland, R., de Jong, E.,

Soeteman, E., Wold, A., Wierenga, E. A. and Kapsenberg, M. L. (2004).

Commensal Gram-negative bacteria prime human dendritic cells for

enhanced IL-23 and IL-27 expression and enhanced Th1 development. Eur.

J. Immunol. 34, 1371�80.

63. Smits, H. H., Engering, A., van der Kleij, D., de Jong, E. C., Schipper, K.,

van Capel, T. M., Zaat, B. A., Yazdanbakhsh, M., Wierenga, E. A., van Kooyk,

Y. and Kapsenberg, M. L. (2005). Selective probiotic bacteria induce

IL-10-producing regulatory T cells in vitro by modulating dendritic cell

function through dendritic cell-specific intercellular adhesion molecule

3-grabbing nonintegrin. J. Allergy Clin. Immunol. 115, 1260�7.

64. Svensson, M., Maroof, A., Ato, M. and Kaye, P. M. (2004). Stromal

cells direct local differentiation of regulatory dendritic cells. Immunity 21,

805�16.

65. Zhang, M., Tang, H., Guo, Z., An, H., Zhu, X., Song, W., Guo, J., Huang, X.,

Chen, T., Wang, J. and Cao, X. (2004). Splenic stroma drives mature

dendritic cells to differentiate into regulatory dendritic cells. Nat. Immunol.

5, 1124�33.

66. Soumelis, V. and Liu, Y. J. (2004). Human thymic stromal lymphopoietin: a

novel epithelial cell-derived cytokine and a potential key player in the

induction of allergic inflammation. Springer Semin. Immunopathol.

25, 325�33.

67. Soumelis, V., Reche, P. A., Kanzler, H., Yuan, W., Edward, G., Homey, B.,

Gilliet, M., Ho, S., Antonenko, S., Lauerma, A. et al. (2002). Human

epithelial cells trigger dendritic cell mediated allergic inflammation by

producing TSLP. Nat. Immunol. 3, 673�80.

68. Rimoldi, M., Chieppa, M., Salucci, V., Avogadri, F., Sonzogni, A.,

Sampietro, G. M., Nespoli, A., Viale, G., Allavena, P. and Rescigno, M.

(2005). Intestinal immune homeostasis is regulated by the crosstalk

between epithelial cells and dendritic cells. Nat. Immunol. 6, 507�14.

69. Kosiewicz, M. M., Nast, C. C., Krishnan, A., Rivera-Nieves, J., Moskaluk,

C. A., Matsumoto, S., Kozaiwa, K. and Cominelli, F. (2001). Th1-type

responses mediate spontaneous ileitis in a novel murine model of Crohn’s

disease. J. Clin. Invest. 107, 695�702.

70. Mastroeni, P. and Menager, N. (2003). Development of acquired immunity

to Salmonella. J. Med. Microbiol. 52, 453�9.

�97

DE

ND

RIT

ICC

EL

LA

CT

IVA

TIO

NA

ND

UP

TA

KE

OF

BA

CT

ER

IAIN

VIV

O

71. Hess, J., Ladel, C., Miko, D. and Kaufmann, S. H. (1996). Salmonella

typhimurium aroA� infection in gene-targeted immunodeficient mice:

major role of CD4þ TCR-alpha beta cells and IFN-gamma in bacterial

clearance independent of intracellular location. J. Immunol. 156, 3321�6.

72. George, A. (1996). Generation of gamma interferon responses in murine

Peyer’s patches following oral immunization. Infect. Immun. 64, 4606�11.

73. Liesenfeld, O., Kosek, J. C. and Suzuki, Y. (1997). Gamma interferon

induces Fas-dependent apoptosis of Peyer’s patch T cells in mice following

peroral infection with Toxoplasma gondii. Infect. Immun. 65, 4682�9.

74. Vossenkamper, A., Struck, D., Alvarado-Esquivel, C., Went, T., Takeda, K.,

Akira, S., Pfeffer, K., Alber, G., Lochner, M., Forster, I. and Liesenfeld, O.

(2004). Both IL-12 and IL-18 contribute to small intestinal Th1-type

immunopathology following oral infection with Toxoplasma gondii, but

IL-12 is dominant over IL-18 in parasite control. Eur. J. Immunol. 34,

3197�207.

�

98

M.

RE

SC

IGN

O

CHAPTER 5

Role of dendritic cells in the innate responseto bacteria

Natalya V. Serbina and Eric G. PamerSloan Kettering Institute

5.1 INTRODUCTION

Innate immunity is an ancient and highly conserved system that provides

the first line of defense upon encounter with pathogenic organisms.

Activation of innate immune responses is a complex process involving

multiple components and distinct steps. The cellular components of

innate immunity include neutrophils, monocytes, macrophages and dendri-

tic cells (DCs). These cells are capable of direct microbicidal activity that

partially depends on inducible nitric synthase (iNOS) and NADPH oxidase

complex that catalyze production of toxic anti-microbial compounds1,2.

Additionally, they secrete a vast array of pro-inflammatory mediators such

as cytokines and chemokines and can recruit and activate other inflam-

matory cells, thus amplifying the immune cascade. Apart from their role

in restricting microbial growth, innate immune responses also provide the

inflammatory context in which adaptive T- and B-cell immune responses

develop.

Dendritic cells are derived from hematopoietic progenitor cells in the

bone marrow and are found in the peripheral circulation as well as in

the lymphoid and non-lymphoid tissues. Dendritic cells can be subdivided

into several subsets based on the expression of the cell surface markers

and different subsets have been ascribed distinct functions during the

immune response3. Since their discovery, dendritic cells have been studied

extensively with regard to their role as antigen-presenting cells4. However,

it is becoming increasingly clear that dendritic cells also play an important

role during the innate immune responses to microbial pathogens.

Dendritic cells are found in peripheral tissues and are strategically

positioned at the sites where pathogen encounters are most frequent,

such as gut, lung and skin. Dendritic cells at these different sites are

morphologically and functionally heterogeneous, potentially optimizing

interactions with pathogens in the context of their local microenvironment.

For example, microbes invading through breaks in the skin encounter

epidermal Langerhans cells and dermal dendritic cells that are highly

phagocytic and can migrate to lymph nodes under inflammatory conditions5.

In the intestinal mucosae, lamina propria dendritic cells express tight

junction proteins and can open junctions between epithelial cells, thereby

enabling sampling of antigens in the intestinal lumen6. Pathogens

entering via the respiratory route are detected by dendritic cells residing

within the epithelium and interstitium of the lung. Microbial contact

generally induces activation of lung dendritic cells and their migration

to lung-draining lymph nodes7.

Dendritic cell uptake of microbes at the epithelial and mucosal sites

and their subsequent migration to lymphoid organs may contribute to

dissemination of microbes throughout the body. During intestinal

Salmonella typhimurium infection, spread of bacteria to extraintestinal sites

requires CD18 expressing phagocytes8. These CD18-positive cells trans-

port Salmonella from intestinal epithelium through the bloodstream to

the spleen. Lung dendritic cells can be infected with Mycobacterium

tuberculosis9 and phagocytose bacteria in vivo, albeit at significantly lower

levels than macrophages10. Migration of infected dendritic cells to lung-

draining lymph nodes has been suggested to trigger development of T cell

immunity as well as contribute to bacterial dissemination.

Dendritic cells express a wide range of TLRs and can recognize and

respond to distinct classes of pathogens11. Additionally, dendritic cells also

express Nod proteins and may be capable of detecting microbes replicating

in cytosol12,13. Infection with live bacteria or stimulation with bacterial

components induces dendritic cells to secrete cytokines and chemokines,

thus directly contributing to the inflammatory milieu. Furthermore,

dendritic cells can orchestrate immune responses by directly activating

other immune cells such as NK cells, T and B cells. Additionally, activated

dendritic cells can secrete nitric oxide and thus may have a direct bacteri-

cidal activity. It was demonstrated that microbial stimulation of dendritic

cells induces very early changes in their gene expression profile suggesting

that they might play a role during the early onset of inflammatory

responses12,13.

Thus, the role of dendritic cells in the innate immune responses is

multifaceted and involves distinct effector functions. Microbial recognition,

in vivo bacterial uptake by dendritic cells and activation of other innate

�

100

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

immune cells are addressed elsewhere in this book. In this chapter, we will

summarize recent advances in our understanding of the role of dendritic

cells in microbial killing, focusing in particular on the murine model

of Listeria monocytogenes infection.

5.2 PATHOGENESIS OF LISTERIA MONOCYTOGENES

Listeria monocytogenes is a pathogenic Gram-positive facultative intra-

cellular bacterium and is the cause of listeriosis. Listeria can infect humans

and risk factors for infection include pregnancy and the immunocom-

promised state that follows chemotherapy or organ transplantation14.

Murine infection with L. monocytogenes has been used extensively as a

model for studying host immune responses to intracellular infection and

has been instrumental in delineating various components of the innate

and adaptive arms of anti-microbial immunity.

The natural route of Listeria infection is via the gastrointestinal tract

when food contaminated with bacteria is ingested. Listeria can infect a variety

of phagocytic and non-phagocytic cells. Invasion of cells of the intestinal

epithelium requires interaction of bacterially expressed internalin A (IntA)

with epithelial E-cadherin molecules15. Another internalin molecule, Int B,

enables bacteria to invade hepatocytes in the liver16. Several receptors have

been shown to interact with IntB such as glycosaminoglycans, hepatocyte

growth factor receptor and complement receptors17�20. Uptake of Listeria

by phagocytic cells can involve macrophage scavenger receptors 21 and

complement receptors 22 on the surface of the host cells.

Inside the infected cells, bacteria are initially found in vacuoles but

rapidly escape into the cytoplasm by secreting membranolytic protein,

listeriolysin O (LLO)23. In some cell types cytoplasmic invasion also

involves the action of bacterially encoded phospholipases24. Expression

of LLO is essential for bacterial virulence and L. monocytogenes strains

deficient in this molecule are highly attenuated. Invasion of the cytosol

by bacteria triggers innate inflammatory responses24,25 and appears to

be required for induction of protective immunity26. In the cytoplasm,

bacteria express the actin-assembly-inducing protein ActA, enabling

bacterial movement through the cytoplasm and into neighboring cells27,28.

ActA is a bacterial virulence factor and ActA-deficient L. monocytogenes

strain are markedly attenuated. Interestingly, while both ActA-deficient

and LLO-deficient strains are attenuated in mice, infection with

ActA-deficient bacteria induces protective long-term immunity29 and, as

noted before, infection with LLO-deficient strain does not26. Thus, protective

�101

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

immunity can be dissociated from virulence. Cytosol invasion by bacteria

establishes the inflammatory environment that optimizes the development

of adaptive T cell responses, potentially by activating dendritic cells.

5.3 KEY MEDIATORS OF INNATE IMMUNE RESPONSES TO

L. MONOCYTOGENES

During L. monocytogenes infection, both innate and adaptive immunity

have been shown to contribute to bacterial clearance. While complete in vivo

clearance of L. monocytogenes is T cell dependent, early control of bacterial

replication depends on induction of innate immunity30,31. Inbred strains

of mice differ with respect to their innate resistance to infection32,33. Genetic

analysis indicates that resistance is a complex genetic trait; several loci

on chromosomes 134, 235,36, 5 and 1334 have been linked to host resistance

against Listeria infection. Following infection, innate immune responses

are triggered rapidly37. The importance of innate immunity is indicated

by the demonstration that mice lacking both CD4 and CD8 T cells are,

for several weeks, able to control infection, although they cannot clear

it in the long term31,38. Innate immune responses to Listeria have

been extensively studied and various immune components have been

delineated. Studies with gene-deficient mice demonstrated that both

TNF-a and IFN-g are crucial for immune protection during infection39,40.

Mice lacking these cytokines or their respective receptors succumb to

infection within a few days, indicating failure of innate immunity41�43.

Although the pivotal role of these cytokines during anti-listerial innate

immune response is established, their cellular sources and their down-

stream effects are not well understood. It is thought that early production

of IFN-g by natural killer cells activates the bactericidal effector functions

of macrophages, thus leading to bacterial killing. However, mice that lack

NK cells are resistant to infection and have normal innate immunity which

suggests that there are NK-independent sources of IFN-g44.Infected macrophages have long been considered to be a major source

of TNF-a. Although the role of TNF-a in the activation and regulation of

innate inflammatory responses is well recognized, its precise mode of

action in defense against Listeria infection remains unresolved. Mice that

express a non-sheddable p55TNFR show innate immune hyperactivity and

are more resistant to L. monocytogenes; however, failure to downregu-

late TNF receptor also leads to inflammatory pathology45. Additionally,

both IL-12 and IL-18 contribute to protection against L. monocytogenes,

possibly through induction of other cytokines46,47. In contrast, mice lacking

�

102

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

Type I interferon receptor or the IRF3 transcription factor have enhanced

resistance to L. monocytogenes infection48�50. In theses studies, signaling

through Type I interferon receptors was suggested to enhance susceptibil-

ity of T cells to apoptosis48,49, down-modulate IL-12 production and

decrease numbers of TNF producing cells50. Thus, induction of innate

immune responses during intracellular bacterial infection is a complex

process involving multiple regulatory steps.

5.4 ROLE OF MYELOID CELLS

Myeloid cells are comprised of distinct subpopulations, all of which

have innate immune functions. Granulocytes, monocytes and dendritic

cell precursors are derived from granulocyte�monocyte progenitor cells

in the bone marrow. During immune responses to bacterial infections,

neutrophils and monocytes play a crucial role. Because of their common

lineage, these cells share number of cell surface receptors and adhesion

molecules such as CD11b, CD11c, Gr-1 and Mac-3 and have overlapping

effector functions. From an immunologist perspective, the common

expression of multiple surface markers makes it difficult to distinguish the

distinct myeloid subpopulations during the innate immune responses.

During innate immune responses to L. monocytogenes, cells of myeloid

lineage play a key role51. The contributions of granulocytes and monocytes

have been characterized in antibody depletion studies. In vivo administration

of RB6-8C5 antibody specific for Gr-1 renders mice highly susceptible

to L. monocytogenes infection52�54. The deleterious effect of RB6-8C5

antibody administration is particularly pronounced when antibody is

given during the first two days of infection53,54. RB6-8C5 antibody reacts

with the common epitope on Ly6G and Ly6C antigens expressed on

monocytes and dendritic cells as well as on neutrophils. Thus, while

neutrophils are undoubtedly indispensable for the early control of bacterial

replication, the potential contribution of other Gr-1-expressing myeloid

cells cannot be discounted.

Administration of a blocking monoclonal antibody specific for type 3

complement receptor (CD11b, Mac-1) impairs immunity of mice to

L. monocytogenes and leads to increased mortality in response to a sublethal

dose of bacteria55. Similar to Gr-1 treatment, anti-CD11b treatment

interferes with immune responses only when given early during infection.

CD11b blocking by antibody leads to a failure to focus monocytic cells at

sites of infection and results in unrestricted bacterial multiplication in

organs, suggesting that CD11b-expressing cells are essential for bacterial

�103

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

containment and killing during early infection. The CD11b-bearing myelo-

monocytic population is comprised of neutrophils, macrophages and

dendritic cells; while all of the cells can contribute to innate immune

responses, their specific contributions to bacterial clearance remain

undefined.

The functions of macrophages and granulocytes during infection have

been extensively studied. It is thought that these cells exert bactericidal

activity by producing reactive nitrogen and oxygen intermediates56 as well

as through the action of phagolysosomal enzymes57. Efficient containment

of L. monocytogenes in the vacuoles of activated macrophages and killing

of bacteria in vitro depends on production of ROI and RNI51,58. Mice

lacking iNOS58,59 and components of the NADPH oxidase complex have

increased susceptibility to L. monocytogenes60,61 suggesting that these

mechanisms contribute to bacterial clearance in vivo.

5.5 TipDCs IN THE INNATE IMMUNE RESPONSES

TO L. MONOCYTOGENES

The direct demonstration that dendritic cells play a role during in vivo

innate immune responses to Listeria came from recent studies that examined

course of infection in mice lacking chemokine receptor CCR2. CCR2 is

expressed on monocytes and dendritic cells and is implicated in the in vivo

migration of these cells under inflammatory conditions62�72. Immunity

to L. monocytogenes is profoundly diminished in CCR2-deficient mice;

knockout mice succumb to infection within 4 days indicating failure of

innate immunity63. Since CCR2 is expressed by circulating monocytes,

it has been suggested that the inability to recruit macrophages to the sites

of infection leads to impaired immune responses in CCR2-deficient mice.

Analysis of innate immune responses in CCR2-deficient mice revealed

markedly diminished levels of TNF and iNOS. It has been postulated that

L. monocytogenes infected macrophages are the major source of these

molecules during infection. Characterization of myeloid populations in the

spleens of infected mice revealed that production of TNF and iNOS was

largely attributable to a novel monocyte-derived dendritic cell population

(TNF/iNOS producing dendritic cells, TipDCs) that is recruited to sites of

bacterial replication in a CCR2-dependent fashion. Phenotypically, TipDCs

express of CD11b, CD11c and high levels of intracellular Mac-3, markers that

are also expressed by some conventional dendritic cells. TipDCs also express

high levels of MHC Class II and co-stimulatory molecules and expression of

these molecules is further induced during the course of infection. In contrast

�

104

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

to conventional dendritic cells, TipDcs do not express CD4, CD8 or CD205.

Interestingly, TipDCs also express Gr-1, a marker expressed by plasmacytoid

dendritic cell subset. However, TipDCs appear to be distinct from plasma-

cytoid population since they do not express B220 and CCR2 knockout

mice have normal levels of Type I IFNs.

To confirm that TNF- and iNOS-producing cells represent a popu-

lation of dendritic cells and not macrophages, their ability to prime T cell

responses was examined. TipDCs induced proliferation of naive allogenic

T cells in in vitro cultures, albeit at lower levels than conventional DCs.

Interestingly, CCR2 deficient mice develop enhanced CD8 T cell responses

(see below) following infection with attenuated strains of L. monocytogenes.

Thus, TipDCs do not appear to contribute to T cell priming in vivo and

have a distinct role during infection.

The essential role of TipDCs in the anti-listerial immune response

is attributed to their ability to produce TNF and nitric oxide during

infection. Intracellular cytokine analysis revealed that TipDCs are capable

of producing copious amounts of TNF in response to stimulation with

Listeria and secrete much more TNF than neutrophils and monocytes.

In the infected spleen, TipDCs appear to be the only major source of

iNOS; consequently, nitric oxide production is drastically reduced in

CCR2-deficient spleens. It remains to be determined whether the ability

of TipDCs to express high levels of iNOS and TNF is due to the unique

expression of receptors or signaling molecules in this subset. Interestingly,

although TipDCs are found in the close proximity to cells harboring

bacteria in vivo, they do not appear to harbor significant numbers of

bacteria in vivo. It is possible that bacteria are rapidly killed and degraded

by TipDCs upon phagocytosis in which case very few cells would appear

directly infected. Alternatively, TipDCs may impact bacterial killing by

orchestrating innate immune responses via production of TNF and nitric

oxide without being directly invaded. NO is a water- and lipid-soluble gas

and it might exert anti-microbial effects by diffusing into infected cells

in the vicinity of Tip-DCs. Additionally, exogenous NO can induce iNOS

expression in infected cells73. In the absence of activation, infection of

macrophages with live L. monocytogenes leads to the rapid destruction of

host cells. Therefore, it may be beneficial to recruit cells capable of sensing

infection and mediating microbicidal effector functions without being

directly infected.

In addition to direct bactericidal activity, nitric oxide production by

TipDCs might have other regulatory functions in the setting of innate

immunity. For example, nitric oxide production has inhibitory effect on

�105

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

T cell proliferation74,75. Interestingly, CD8 T cell responses in the spleens

of CCR2-deficient mice are larger than those observed in the wild type

spleens; thus, TipDCs might regulate the magnitude of developing T cell

responses.

The CCR2-expressing monocytes circulating in the blood of naive

mice and recruited to the sites of inflammation via CCR2 have recently

been described69. After migration to the inflamed tissues, these cells

have a capacity to differentiate into dendritic cells. Similar to TipDCs, the

inflammatory monocytes express Gr-1 and CD11b and do not express

CD4 and CD8 molecules. Thus, it is conceivable that these circulating cells

represent precursors of TipDCs.

TNF secretion by TipDCs requires MyD88-mediated signaling but is

intact in the absence of TLR225. It is possible that the combination of

multiple distinct TLRs and/or the levels of receptor expression determine

the capacity of TipDCs to produce these inflammatory mediators.

It remains to be determined whether TipDCs play a role during immune

responses to other bacterial pathogens. In vitro infection of dendritic cells

with M. tuberculosis and S. typhimurium induces iNOS expression and

secretion of nitric oxide76�79. Recently, recruitment of CCR2þCD11cint

dendritic cells to the lungs of mice infected with M. tuberculosis has

been reported70. TNF and iNOS production by lung CD11c intermediate

cells was not examined in this study and it remains to be determined whether

they represent a TipDC population. Another study reported the presence

of monocyte-derived dendritic cells capable of bactericidal activity against

intracellular M. tuberculosis in bronchoalveolar lavage samples80. At this

point, further studies are needed to determine whether recruitment of

TipDCs represents a common mechanism for defense against intracellular

bacterial infections.

5.6 DENDRITIC CELL INTERACTION WITH L. MONOCYTOGENES

Much of what is known regarding dendritic cell interaction with pathogens

has been learned from in vitro studies. Human and murine dendritic cells

can be derived by culturing blood or bone marrow monocytes with GM-CSF

in the presence or absence of IL-4. The resulting cell populations express

high levels of MHC Class II and co-stimulatory molecules. GM-CSF

differentiated cells have been very instrumental in examining dendritic cell

interactions with T cells and impact of bacterial infection on this cell type.

Human and murine dendritic cells can be infected with L. monocyto-

genes in vitro81�83. Bacterial internalization occurs independent of

�

106

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

internalin A and B and involves glycosylated receptors on the surface of

dendritic cells81. Whether Listeria escape vacuoles and replicate in the

cytoplasm of infected dendritic cells is controversial with reports demon-

strating bacterial escape into cytoplasm of murine dendritic cells82,83, while

another study reported that internalized Listeria are contained predomi-

nantly in phagosomes81. Interestingly, although bacteria induce killing of

murine dendritic cells, a small proportion of infected cells remains infected

for a prolonged period of time83. In other infectious disease settings, the

ability of dendritic cells to harbor live bacteria for a prolonged period of

time has been suggested to contribute to bacterial dissemination and T cell

priming80,84�86. Invasion of immature dendritic cells by L. monocytogenes

induces their maturation and enhanced expression of MHC Class II and

co-stimulatory molecules81,87; in one study this effect appeared to be

largely due to listerial lipoteichoic acid81. In addition to inducing dendritic

cell maturation, infection also stimulates in vitro cytokine production by

DCs88,89.

Activation of the innate immune cells and subsequent cytokine

secretion occurs following triggering of the pathogen-recognition receptors,

Toll-like receptors (TLRs, see Chapter 2). Various members of the TLR

family recognize evolutionary conserved, pathogen-derived components

such as lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid

(LTA), CpG DNA, bacterial flagellin and viral double-stranded RNA. TLR

family members signal via the MyD88 adaptor molecule90. Signaling

through TLR-MyD88 pathway is critical for initiation of antimicrobial

immune responses and Myd88-deficient mice are highly susceptible to

a number of bacterial pathogens. Immune responses to L. monocytogenes

have been examined in mice lacking specific innate immune receptors as

well as MyD88. Genetic deletion of Myd88 in mice leads to dramatically

diminished resistance to L. monocytogenes91,92. Moreover, MyD88-deficient

mice display greater susceptibility than mice lacking IFN-g or both IL-12

and IFN-g91 IL-1 and IL-18-signaling is MyD88-dependent93. Impaired

immunity in the absence of MyD88 is attributed to loss of TLR-mediated

signals since mice deficient in Caspase-1 (and therefore secreted forms

of IL-1 and IL-18) are only marginally susceptible to Listeria92,94.

Listeria can be recognized through distinct TLRs such as TLR2,

receptor for petidoglycan, lipoproteins and lipoteichoic acid, TLR5, receptor

for flagellin and TLR9, receptor for unmethylated CpG DNA. Infection

with L. monocytogenes induces IL-12 and IL-18 secretion by human DCs109

while stimulation with listerial LTA induced secretion of IL-18 but only

minimal IL-12, suggesting that live bacteria are recognized by multiple TLRs.

�107

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

Recognition of L. monocytogenes through TLR2 and TLR5 has

been documented91,95. However, conflicting results have been reported

regarding the role of TLR2 in innate immune responses to L. monocytogenes

infection25,91,92 and in vivo data on TLR5 is still lacking. Recently,

administration of immunostimulatory CpG oligodeoxynucleotides was

shown to render newborn mice resistant to L. monocytogenes infection.

In CpG-treated mice, dendritic cells, macrophages and B cells responded

to stimulation by secreting IFN-g, TNF-a and IL-1296. It remains to be

established whether TLR9-mediated recognition of Listeria contributes

to the immune responses in vivo.

Dendritic cell maturation and production of IL-12, IL-10, IL-6 and

TNF-a is efficiently triggered by cytosol-invasive and not LLO-deficient

bacteria indicating that dendritic cells are capable of sensing the presence

of Listeria in the cytoplasm87. Recently, microbial recognition by cyto-

plasmic proteins Nod1 and Nod2 has been reported. Nod1 and Nod2

belong to NBS-LRR family of mammalian proteins and recognize bacterial

petidoglycan motifs associated with Gram-positive and Gram-negative cell

wall. Nod2 recognizes muramyl dipeptide (MDP), a conserved structure

present in the cell wall of Gram-positive and Gram-negative bacteria97�99.

Nod 1 detects a unique diaminopimelate-containing N-acetylglucosamine-

N-acetylmuramic acid tripeptide motif present in the Gram-negative

bacterial peptidoglycan100. Dendritic cells express Nod1 and Nod2 and

release cytokines in response to stimulation with muropeptides12,13. Nod2-

deficient mice have increased susceptibility to L. monocytogenes admin-

istered via the intragastric route101, suggesting that Listeria-derived cell

wall components are recognized by Nod2 in vivo. Additionally, the cell

wall of L. monocytogenes contains mesoDAP and thus can potentially be

recognized by Nod1. Although, extracts of L. monocytogenes do not induce

Nod1-mediated signaling in vitro98, the potential role of Nod1 recognition

during in vivo infection cannot be ruled out. Both Nod1 and Nod2 associate

with the serine/threonine kinase RIP2 and their signaling leads to NF-kB

activation. The role of Rip2 in defense against L. monocytogenes has been

examined. IL-6 production by L. monocytogenes infected macrophages

is diminished in the absence of Rip2 and Rip2-deficient mice are more

susceptible to in vivo infection102,103. Rip2 is positioned downstream of

TLR and Nod proteins in the signaling cascade. However, MyD88-deficient

mice are significantly more susceptible to Listeria infection than Rip2-

deficient mice, suggesting that Rip2 plays a more limited role in the TLR-

mediated immune activation. In vitro infection of cells with L. monocytogenes

induces NF-kB activation104, production of type I IFNs24, and MCP-125 that is

�

108

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

dependent on bacterial localization to cytoplasm. A recent report demon-

strated that secretion of type I IFNs induced by cytoplasmic bacteria requires

serine-threonine kinase TNFR-associated NF-kB kinase (TANK-binding

kinase 1 (TBK1))105. Further studies are required to address the contribution

of these signaling molecules to the in vivo activation of dendritic cells and

macrophages during innate immune responses to L. monocytogenes.

5.7 IN VIVO FUNCTION OF DENDRITIC CELLS

In lymphoid organs, dendritic cells can be divided into CD8-positive and

CD8-negative populations; the CD8-negative population can be further

subdivided based on their expression of CD4 and CD2053. CD8-positive

dendritic cells express CD11c, CD205 and high levels of MHC Class II

and do not express CD11b. CD8-negative dendritic cell subsets express

varied levels of CD11b and Gr-1. Due to the fact that many cell surface

markers are shared between dendritic cell subsets and other cells of

myeloid lineage, the contribution of distinct subpopulations to immune

responses and bacterial clearance in vivo is difficult to assess.

Dendritic cells are infected with L. monocytogenes in vivo after intestinal

inoculation106. Listeria-loaded dendritic cells are first detected beneath the

epithelial lining of Peyer’s patches; 6 hours following infection, bacteria-

containing cells are present in the draining lymph nodes suggesting

that dendritic cells may be involved in bacterial dissemination. The role

of dendritic cells during in vivo L. monocytogenes infection has been

addressed in mice treated with Flt-3 ligand, a hemapoietic growth factor

that drastically increases the numbers of DCs in organs. Treatment of

neonatal mice with Flt-3 ligand increases the numbers of CD11cþMHC

Class IIhigh and CD11cþB220þ dendritic cells in the spleen, liver, skin

and peritoneum and enhances resistance to L. monocytogenes in IL-12-

dependent manner suggesting that dendritic cells play an active part during

innate immune responses107. However, different results were reported by

another study in which Flt-3 ligand was administered to adult mice and

this treatment led to impaired protective immunity despite enhanced T cell

priming110. Although these studies provide the insight into our under-

standing of potential functions of dendritic cells, they are based on

the artificial enhancement of dendritic cell numbers and may not reflect

the immune responses following infection in vivo.

Recently, the in vivo role of dendritic cells in the priming of adaptive

immune responses has been examined using transient in vivo depletion

of CD11c-expressing cells108. Utilizing diphtheria toxin-based system,

�109

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

Jung et al. demonstrated that short-term depletion of CD11c-expressing

dendritic cells abrogates in vivo CD8 T cell priming during L. monocytogenes

infection. It remains to be determined whether T cell priming by dendritic

cells during infection is carried out by a small fraction of cells that are

directly infected or whether it involves antigen cross-presentation. This

study reinforced the idea that potent in vitro antigen-presenting capacity

of dendritic cells can be directly translated to their in vivo role. The impact

of CD11cþ cell depletion on the development of innate immune responses

to L. monocytogenes remains unclear.

5.8 CONCLUSIONS

In the past two decades, research in the dendritic cell field has been

centered on the antigen presenting capacity of these cells. However, it is

becoming increasingly clear that dendritic cells contribute to many aspects

of innate inflammatory responses. Understanding the molecular and

cellular mechanisms of dendritic cell functions during innate immune

responses is important for better understanding of the pathogenesis of

infectious diseases as well as immune-related disorders and will lead to

the development of novel immunologic therapies.

REFERENCES

1. Taylor, P. R. et al. (2005). Macrophage receptors and immune recognition.

Annu. Rev. Immunol. 23, 901�44.

2. Segal, A. W. (2005). How neutrophils kill microbes. Annu. Rev. Immunol.

23, 197�223.

3. Shortman, K. and Y. J. Liu (2002). Mouse and human dendritic cell

subtypes. Nat. Rev. Immunol. 2(3), 151�61.

4. Steinman, R. M. (1991). The dendritic cell system and its role in

immunogenicity. Annu. Rev. Immunol. 9, 271�96.

5. Carbone, F. R., G. T. Belz, and W. R. Heath (2004). Transfer of antigen

between migrating and lymph node-resident DCs in peripheral T-cell

tolerance and immunity. Trends Immunol. 25(12), 655�8.

6. Rescigno, M. et al. (2001). Dendritic cells express tight junction proteins

and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol.

2(4), 361�7.

7. Lambrecht, B. N., J. B. Prins, and H. C. Hoogsteden (2001). Lung dendritic

cells and host immunity to infection. Eur. Respir. J. 18(4), 692�704.

�

110

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

8. Vazquez-Torres, A. et al. (1999). Extraintestinal dissemination of Salmonella

by CD18-expressing phagocytes. Nature 401(6755), 804�8.

9. Gonzalez-Juarrero, M. and I. M. Orme (2001). Characterization of

murine lung dendritic cells infected with Mycobacterium tuberculosis.

Infect. Immun. 69(2), 1127�33.

10. Pedroza-Gonzalez, A. et al. (2004). In situ analysis of lung antigen-

presenting cells during murine pulmonary infection with

virulent Mycobacterium tuberculosis. Int. J. Exp. Pathol. 85(3), 135�45.

11. Iwasaki, A. and R. Medzhitov (2004). Toll-like receptor control of the

adaptive immune responses. Nat. Immunol. 5(10), 987�95.

12. Fritz, J. H. et al. (2005). Synergistic stimulation of human monocytes

and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-

activating agonists. Eur. J. Immunol. 35(8), 2459�70.

13. Gutierrez, O. et al. (2002). Induction of Nod2 in myelomonocytic

and intestinal epithelial cells via nuclear factor-kappa B activation. J. Biol.

Chem. 277(44), 41701�5.

14. Gellin, B. G. and C. V. Broome (1989). Listeriosis. JAMA 261(9), 1313�20.

15. Gaillard, J. L. et al. (1991). Entry of L. monocytogenes into cells is

mediated by internalin, a repeat protein reminiscent of surface antigens

from gram-positive cocci. Cell 65(7), 1127�41.

16. Dramsi, S. et al. (1995). Entry of Listeria monocytogenes into hepatocytes

requires expression of inIB, a surface protein of the internalin multigene

family. Mol. Microbiol. 16(2), 251�61.

17. Braun, L., B. Ghebrehiwet, and P. Cossart (2000). gC1q-R/p32, a

C1q-binding protein, is a receptor for the InlB invasion protein of Listeria

monocytogenes. EMBO J. 19(7), 1458�66.

18. Shen, Y. et al. (2000). InIB-dependent internalization of Listeria is mediated

by the Met receptor tyrosine kinase. Cell 103(3), 501�10.

19. Marino, M. et al. (2002). GW domains of the Listeria monocytogenes invasion

protein InlB are SH3-like and mediate binding to host ligands. EMBO J.

21(21), 5623�34.

20. Jonquieres, R., J. Pizarro-Cerda, and P. Cossart (2001). Synergy between the

N- and C-terminal domains of InlB for efficient invasion of non-phagocytic

cells by Listeria monocytogenes. Mol. Microbiol. 42(4), 955�65.

21. Dunne, D. W. et al. (1994). The type I macrophage scavenger receptor

binds to Gram-positive bacteria and recognizes lipoteichoic acid. Proc.

Natl Acad. Sci. U SA 91(5), 1863�7.

22. Drevets, D. A. and P. A. Campbell (1991). Roles of complement and

complement receptor type 3 in phagocytosis of Listeria monocytogenes by

�111

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

inflammatory mouse peritoneal macrophages. Infect. Immun. 59(8),

2645�52.

23. Bielecki, J. et al. (1990). Bacillus subtilis expressing a haemolysin gene

from Listeria monocytogenes can grow in mammalian cells. Nature

345(6271), 175�6.

24. O’Riordan, M. et al. (2002). Innate recognition of bacteria by a macrophage

cytosolic surveillance pathway. Proc. Natl Acad. Sci. U SA 99(21), 13861�6.

25. Serbina, N. V. et al. (2003). Sequential MyD88-independent

and -dependent activation of innate immune responses to intracellular

bacterial infection. Immunity 19(6), 891�901.

26. Berche, P., J. L. Gaillard, and P. J. Sansonetti (1987). Intracellular

growth of Listeria monocytogenes as a prerequisite for in vivo induction of

T cell-mediated immunity. J. Immunol. 138(7), 2266�71.

27. Domann, E. et al. (1992). A novel bacterial virulence gene in

Listeria monocytogenes required for host cell microfilament interaction

with homology to the proline-rich region of vinculin. EMBO J. 11(5),

1981�90.

28. Kocks, C. et al. (1992). L. monocytogenes-induced actin assembly requires

the actA gene product, a surface protein. Cell 68(3), 521�31.

29. Goossens, P. L. and G. Milon (1992). Induction of protective CD8þ

T lymphocytes by an attenuated Listeria monocytogenes actA mutant.

Int. Immunol. 4(12), 1413�18.

30. Harty, J. T. and M. J. Bevan (1996). CD8 T-cell recognition of macrophages

and hepatocytes results in immunity to Listeria monocytogenes. Infect.

Immun. 64(9), 3632�40.

31. Bancroft, G. J., R. D. Schreiber, and E. R. Unanue (1991). Natural

immunity: a T-cell-independent pathway of macrophage activation,

defined in the scid mouse. Immunol. Rev. 124, 5�24.

32. Stevenson, M. M., P. A. Kongshavn, and E. Skamene (1981). Genetic

linkage of resistance to Listeria monocytogenes with macrophage

inflammatory responses. J. Immunol. 127(2), 402�7.

33. Cheers, C. and I. F. McKenzie (1978). Resistance and susceptibility

of mice to bacterial infection: genetics of listeriosis. Infect. Immun. 19(3),

755�62.

34. Boyartchuk, V. et al. (2004). The host resistance locus sst1 controls

innate immunity to Listeria monocytogenes infection in immunodeficient

mice. J. Immunol. 173(8), 5112�20.

35. Gervais, F., C. Desforges, and E. Skamene (1989). The C5-sufficient

A/J congenic mouse strain. Inflammatory response and resistance to

Listeria monocytogenes. J. Immunol. 142(6), 2057�60.

�

112

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

36. Gervais, F., M. Stevenson, and E. Skamene (1984). Genetic control of

resistance to Listeria monocytogenes: regulation of leukocyte inflammatory

responses by the Hc locus. J. Immunol. 132(4), 2078�83.

37. Unanue, E. R. (1997). Studies in listeriosis show the strong symbiosis

between the innate cellular system and the T-cell response. Immunol. Rev.

158, 11�25.

38. Nickol, A. D. and P. F. Bonventre (1977). Anomalous high native

resistance to athymic mice to bacterial pathogens. Infect. Immun. 18(3),

636�45.

39. Buchmeier, N. A. and R. D. Schreiber (1985). Requirement of endogenous

interferon-gamma production for resolution of Listeria monocytogenes

infection. Proc. Natl Acad. Sci. U S A 82(21), 7404�8.

40. Havell, E. A. (1989). Evidence that tumor necrosis factor has an

important role in antibacterial resistance. J. Immunol. 143(9), 2894�9.

41. Pfeffer, K. et al. (1993). Mice deficient for the 55 kd tumor necrosis

factor receptor are resistant to endotoxic shock, yet succumb to

L. monocytogenes infection. Cell 73(3), 457�67.

42. Rothe, J. et al. (1993). Mice lacking the tumour necrosis factor

receptor 1 are resistant to TNF-mediated toxicity but highly

susceptible to infection by Listeria monocytogenes. Nature 364(6440),

798�802.

43. Harty, J. T. and M. J. Bevan (1995). Specific immunity to Listeria mono-

cytogenes in the absence of IFN gamma. Immunity 3(1), 109�17.

44. Andersson, A. et al. (1998). Early IFN-gamma production and innate

immunity during Listeria monocytogenes infection in the absence of NK

cells. J. Immunol. 161(10), 5600�6.

45. Xanthoulea, S. et al. (2004). Tumor necrosis factor (TNF) receptor shedding

controls thresholds of innate immune activation that balance opposing

TNF functions in infectious and inflammatory diseases. J. Exp.Med. 200(3),

367�76.

46. Tripp, C. S. et al. (1994). Neutralization of IL-12 decreases resistance to

Listeria in SCID and C.B-17 mice. Reversal by IFN-gamma. J. Immunol.

152(4), 1883�7.

47. Neighbors, M. et al. (2001). A critical role for interleukin 18 in primary

and memory effector responses to Listeria monocytogenes that extends

beyond its effects on interferon gamma production. J. Exp. Med. 194(3),

343�54.

48. Carrero, J. A., B. Calderon, and E. R. Unanue (2004). Type I interferon

sensitizes lymphocytes to apoptosis and reduces resistance to listeria

infection. J. Exp. Med. 200(4), 535�40.

�113

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

49. O’Connell, R. M. et al. (2004). Type I interferon production enhances

susceptibility to Listeria monocytogenes infection. J. Exp. Med. 200(4),

437�45.

50. Auerbuch, V. et al. (2004). Mice lacking the type I interferon receptor

are resistant to Listeria monocytogenes. J. Exp. Med. 200(4), 527�33.

51. Beckerman, K. P. et al. (1993). Release of nitric oxide during the

T cell-independent pathway of macrophage activation. Its role

in resistance to Listeria monocytogenes. J. Immunol. 150(3), 888�95.

52. Conlan, J. W. and R. J. North (1994). Neutrophils are essential for early anti-

Listeria defense in the liver, but not in the spleen or peritoneal cavity,

as revealed by a granulocyte-depleting monoclonal antibody. J. Exp. Med.

179(1), 259�68.

53. Rogers, H. W. and E. R. Unanue (1993). Neutrophils are involved in

acute, nonspecific resistance to Listeria monocytogenes in mice. Infect.

Immun. 61(12), 5090�6.

54. Czuprynski, C. J. et al. (1994). Administration of anti-granulocyte mAb

RB6-8C5 impairs the resistance of mice to Listeria monocytogenes infection.

J. Immunol. 152(4), 1836�46.

55. Rosen, H., S. Gordon, and R. J. North (1989). Exacerbation of murine

listeriosis by a monoclonal antibody specific for the type 3 complement

receptor of myelomonocytic cells. Absence of monocytes at infective

foci allows Listeria to multiply in nonphagocytic cells. J. Exp. Med. 170(1),

27�37.

56. Fang, F. C. (2004). Antimicrobial reactive oxygen and nitrogen species:

concepts and controversies. Nat. Rev. Microbiol. 2(10), 820�32.

57. Amer, A. O. and M. S. Swanson (2002). A phagosome of one’s own:

a microbial guide to life in the macrophage. Curr. Opin. Microbiol. 5(1),

56�61.

58. Shiloh, M. U. et al. (1999). Phenotype of mice and macrophages deficient in

both phagocyte oxidase and inducible nitric oxide synthase. Immunity

10(1), 29�38.

59. Serbina, N. V. et al. (2003). TNF/iNOS-producing dendritic cells mediate

innate immune defense against bacterial infection. Immunity 19(1), 59�70.

60. Endres, R. et al. (1997). Listeriosis in p47(phox�/�) and TRp55�/� mice:

protection despite absence of ROI and susceptibility despite presence of

RNI. Immunity 7(3), 419�32.

61. Dinauer, M. C., M. B. Deck, and E. R. Unanue (1997). Mice lacking reduced

nicotinamide adenine dinucleotide phosphate oxidase activity show

increased susceptibility to early infection with Listeria monocytogenes.

J. Immunol. 158(12), 5581�3.

�

114

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

62. Kuziel, W. A. et al. (1997). Severe reduction in leukocyte adhesion and

monocyte extravasation in mice deficient in CC chemokine receptor 2.

Proc. Natl Acad. Sci. U S A 94(22), 12053�8.

63. Kurihara, T. et al. (1997). Defects in macrophage recruitment and

host defense in mice lacking the CCR2 chemokine receptor. J. Exp. Med.

186(10), 1757�62.

64. Sato, N. et al. (2000). CC chemokine receptor (CCR)2 is required

for Langerhans cell migration and localization of T helper cell type 1

(Th1)-inducing dendritic cells. Absence of CCR2 shifts the Leishmania

major-resistant phenotype to a susceptible state dominated by Th2

cytokines, b cell outgrowth, and sustained neutrophilic inflammation.

J. Exp. Med. 192(2), 205�18.

65. Izikson, L. et al. (2000). Resistance to experimental autoimmune encepha-

lomyelitis in mice lacking the CC chemokine receptor (CCR)2. J. Exp. Med.

192(7), 1075�80.

66. Vecchi, A. et al. (1999). Differential responsiveness to constitutive vs.

inducible chemokines of immature and mature mouse dendritic cells.

J. Leukoc. Biol. 66(3), 489�94.

67. Mack, M. et al. (2001). Expression and characterization of the chemokine

receptors CCR2 and CCR5 in mice. J. Immunol. 166(7), 4697�704.

68. Merad, M. et al. (2002). Langerhans cells renew in the skin throughout

life under steady-state conditions. Nat. Immunol. 3(12), 1135�41.

69. Geissmann, F., S. Jung, and D. R. Littman (2003). Blood monocytes

consist of two principal subsets with distinct migratory properties.

Immunity 19(1), 71�82.

70. Peters, W. et al. (2004). CCR2-dependent trafficking of F4/80dim macro-

phages and CD11cdim/intermediate dendritic cells is crucial for T cell

recruitment to lungs infected with Mycobacterium tuberculosis. J. Immunol.

172(12), 7647�53.

71. Kurihara, T. and R. Bravo (1996). Cloning and functional expression

of mCCR2, a murine receptor for the C�C chemokines JE and FIC. J. Biol.

Chem. 271(20), 11603�7.

72. Sozzani, S. et al. (1997). Receptor expression and responsiveness of human

dendritic cells to a defined set of CC and CXC chemokines. J. Immunol.

159(4), 1993�2000.

73. Sheffler, L. A. et al. (1995). Exogenous nitric oxide regulates IFN-gamma

plus lipopolysaccharide-induced nitric oxide synthase expression in mouse

macrophages. J. Immunol. 155(2), 886�94.

74. Lu, L. et al. (1996). Induction of nitric oxide synthase in mouse

dendritic cells by IFN-gamma, endotoxin, and interaction with allogeneic

�115

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

T cells: nitric oxide production is associated with dendritic cell apoptosis.

J. Immunol. 157(8), 3577�86.

75. Albina, J. E. and W. L. Henry, Jr. (1991). Suppression of lymphocyte

proliferation through the nitric oxide synthesizing pathway. J. Surg. Res.

50(4), 403�9.

76. Eriksson, S., B. J. Chambers, and M. Rhen (2003). Nitric oxide produced

by murine dendritic cells is cytotoxic for intracellular Salmonella enterica sv.

Typhimurium. Scand. J. Immunol. 58(5), 493�502.

77. Cheminay, C., A. Mohlenbrink, and M. Hensel (2005). Intracellular

Salmonella inhibit antigen presentation by dendritic cells. J. Immunol.

174(5), 2892�9.

78. Fremond, C. M. et al. (2004). Fatal Mycobacterium tuberculosis infection

despite adaptive immune response in the absence of MyD88. J. Clin. Invest.

114(12), 1790�9.

79. Bodnar, K. A., N. V. Serbina, and J. L. Flynn (2001). Fate of

Mycobacterium tuberculosis within murine dendritic cells. Infect. Immun.

69(2), 800�9.

80. Buettner, M. et al. (2005). Inverse correlation of maturity and

antibacterial activity in human dendritic cells. J. Immunol. 174(7), 4203�9.

81. Kolb-Maurer, A. et al. (2000). Listeria monocytogenes-infected human

dendritic cells: uptake and host cell response. Infect. Immun. 68(6),

3680�8.

82. Paschen, A. et al. (2000). Human dendritic cells infected by Listeria

monocytogenes: induction of maturation, requirements for phagolysoso-

mal escape and antigen presentation capacity. Eur. J. Immunol. 30(12),

3447�56.

83. Guzman, C. A. et al. (1995). Interaction of Listeria monocytogenes with

mouse dendritic cells. Infect. Immun. 63(9), 3665�73.

84. Neild, A. L. and C. R. Roy (2003). Legionella reveal dendritic cell functions

that facilitate selection of antigens for MHC class II presentation.

Immunity 18(6), 813�23.

85. Macpherson, A. J. and T. Uhr (2004). Induction of protective IgA by

intestinal dendritic cells carrying commensal bacteria. Science 303(5664),

1662�5.

86. Hopkins, S. A. et al. (2000). A recombinant Salmonella typhimurium

vaccine strain is taken up and survives within murine Peyer’s patch

dendritic cells. Cell Microbiol. 2(1), 59�68.

87. Brzoza, K. L., A. B. Rockel, and E. M. Hiltbold (2004). Cytoplasmic entry

of Listeria monocytogenes enhances dendritic cell maturation and T cell

differentiation and function. J. Immunol. 173(4), 2641�51.

�

116

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

88. Feng, H. et al. (2005). Listeria-infected myeloid dendritic cells produce

IFN-beta, priming T cell activation. J. Immunol. 175(1), 421�32.

89. Kolb-Maurer, A. et al. (2003). Production of IL-12 and IL-18 in human

dendritic cells upon infection by Listeria monocytogenes. FEMS Immunol.

Med. Microbiol. 35(3), 255�62.

90. Takeda, K. and S. Akira (2004). TLR signaling pathways. Semin. Immunol.

16(1), 3�9.

91. Seki, E. et al. (2002). Critical roles of myeloid differentiation factor

88-dependent proinflammatory cytokine release in early phase clearance

of Listeria monocytogenes in mice. J. Immunol. 169(7), 3863�8.

92. Edelson, B. T. and E. R. Unanue (2002). MyD88-dependent but Toll-like

receptor 2-independent innate immunity to Listeria: no role for either in

macrophage listericidal activity. J. Immunol. 169(7), 3869�75.

93. Adachi, O. et al. (1998). Targeted disruption of the MyD88 gene results

in loss of IL-1- and IL-18-mediated function. Immunity 9(1), 143�50.

94. Tsuji, N. M. et al. (2004). Roles of caspase-1 in Listeria infection in

mice. Int. Immunol. 16(2), 335�43.

95. Hayashi, F. et al. (2001). The innate immune response to bacterial

flagellin is mediated by Toll-like receptor 5. Nature 410(6832), 1099�103.

96. Ito, S. et al. (2005). CpG oligodeoxynucleotides enhance neonatal resistance

to Listeria infection. J. Immunol. 174(2), 777�82.

97. Inohara, N. et al. (2003). Host recognition of bacterial muramyl

dipeptide mediated through NOD2. Implications for Crohn’s disease.

J. Biol. Chem. 278(8), 5509�12.

98. Girardin, S. E. et al. (2003). Nod2 is a general sensor of peptidoglycan

through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278(11),

8869�72.

99. Li, J. et al. (2004). Regulation of IL-8 and IL-1beta expression in Crohn’s

disease associated NOD2/CARD15 mutations. Hum. Mol. Genet. 13(16),

1715�25.

100. Girardin, S. E. et al. (2003). Nod1 detects a unique muropeptide from

Gram-negative bacterial peptidoglycan. Science 300(5625), 1584�7.

101. Kobayashi, K. S. et al. (2005). Nod2-dependent regulation of

innate and adaptive immunity in the intestinal tract. Science 307(5710),

731�4.

102. Chin, A. I. et al. (2002). Involvement of receptor-interacting protein 2

in innate and adaptive immune responses. Nature 416(6877), 190�4.

103. Kobayashi, K. et al. (2002). RICK/Rip2/CARDIAK mediates signalling

for receptors of the innate and adaptive immune systems. Nature

416(6877), 194�9.

�117

RO

LE

OF

DE

ND

RIT

ICC

EL

LS

INT

HE

INN

AT

ER

ES

PO

NS

ET

OB

AC

TE

RIA

104. Hauf, N. et al. (1997). Listeria monocytogenes infection of P388D1 macro-

phages results in a biphasic NF-kappaB (RelA/p50) activation induced

by lipoteichoic acid and bacterial phospholipases and mediated

by IkappaBalpha and IkappaBbeta degradation. Proc. Natl Acad. Sci. U SA

94(17), 9394�9.

105. O’Connell, R. M. et al. (2005). Immune activation of type I IFNs by Listeria

monocytogenes occurs independently of TLR4, TLR2, and receptor

interacting protein 2 but involves TNFR-associated NF kappa

B kinase-binding kinase 1. J. Immunol. 174(3), 1602�7.

106. Pron, B. et al. (2001). Dendritic cells are early cellular targets of Listeria

monocytogenes after intestinal delivery and are involved in bacterial

spread in the host. Cell Microbiol. 3(5), 331�40.

107. Vollstedt, S. et al. (2003). Flt3 ligand-treated neonatal mice have

increased innate immunity against intracellular pathogens and efficiently

control virus infections. J. Exp. Med. 197(5), 575�84.

108. Jung, S. et al. (2002). In vivo depletion of CD11c(þ) dendritic cells

abrogates priming of CD8(þ) T cells by exogenous cell-associated anti-

gens. Immunity 17(2), 211�20.

109. Kolb-Maurer, A. et al. (2003). Induction of IL-12 and IL-18 in human

dendritic cells upon infection by Listeria monocytogenes. FEMS Immunol.

Med. Microbiol. 35(3), 255�62.

110. Alanir, R. C. et al. (2004). Increased dendritic cell numbers impair

protective immunity to intracellular bacteria despite augmenting

antigen-specific CD8þ T lymphocyte responses. J. Immunol. 172, 3725�35.

�

118

N.

V.

SE

RB

INA

AN

DE.

G.

PA

ME

R

CHAPTER 6

Interactions between natural killer anddendritic cells during bacterial infections

Guido FerlazzoIstituto Nazionale Ricerca sul Cancro and University of Messina

Natural killer (NK) cells represent a distinct lymphoid population

characterized by unique phenotypic and functional features. NK cells

were originally identified on a functional basis as this denomination was

assigned to lymphoid cells capable of lysing tumor cell lines in the absence

of prior stimulation in vivo or in vitro1. Both their origin and the

mechanism(s) mediating their function remained mysterious until recently.

Regarding their origin, it has been shown that NK cells derive from

a precursor common to T cells and expressing the CD34þCD7þ phenotype.

In addition, functional NK cells can be obtained in vitro and in vivo

from (CD34þ) haematopoietic precursors isolated from several different

sources2–6. The cell maturation in vitro has been shown to require

appropriate feeder cells and/or IL-15. The molecular mechanisms under-

lying the ability of NK cells to discriminate between normal and tumor cells,

predicted by the ‘‘missing self hypothesis’’7. have been clarified only

during the past decade. It has been shown that NK cells recognize MHC-

class I molecules through surface receptors delivering inhibitory, rather

than activating, signals. Accordingly, NK cells lyse target cells that have

lost (or express low amounts of) MHC class I molecules. This event

occurs frequently in tumors or in cells infected by some viruses such

as certain herpesviruses or adenoviruses. In addition to providing a first

line of defence against viruses, NK cells release various cytokines and

chemokines. These released cytokines can control bacterial spreading

but also induce or modulate inflammatory responses, hematopoiesis, and

control the growth and function of monocytes and granulocytes. Finally,

the functional links between NK and dendritic cells (DCs) have

been widely investigated in recent years and different studies have

demonstrated that reciprocal activations ensue upon NK/DC interactions.

More recently, the anatomical sites where these interactions take place

have been identified together with the related cell subsets involved8.

Remarkably, there is now ‘‘in vivo’’ evidence that this cellular cross-talk

occurring during the innate phase of the immune response against

bacteria or bacterial products can deeply affect the magnitude and the

quality of the subsequent adaptive response9.

These new experimental evidences emphasize the relevance of the

interplay between DCs and NK cells during bacterial infections.

6.1 NK CELLS EVOLVED TO COOPERATE WITH THE

ADAPTIVE IMMUNITY

Thus, NK cells are not merely cytotoxic lymphocytes competent in con-

taining viral and tumor spreading but can now rather be considered as

crucial fine-tuning effector cells widely involved in different phases of the

immune response.

Indeed, while NK cells have long been defined as ‘‘primitive’’ and ‘‘non-

specific’’ effector cells, we have now a different perception of these cells.

First, it is evident that NK cells evolved to adapt to and cooperate with

mechanisms of the specific immunity: they have evolved receptors for

the Fc portion of IgG that allow killing of antibody-coated target cells

or certain pathogens; they also release a number of cytokines that

regulate T cell activation and function. Importantly, an early activation of

NK cells during immune responses may influence the quality of the

subsequent T cell response by inducing a Th1 polarization. Second, NK

cells have evolved a mechanism allowing the rapid detection and killing

of potentially dangerous cells characterized by an altered expression of

MHC class I antigens due to infections. Human NK cells have been

shown to express different human leukocyte antigen (HLA)-class I-specific

inhibitory receptors. A family of these receptors (termed killer Ig-like

receptors (KIR)) detect shared allelic determinants of HLA class I molecules

while others display a more ‘‘promiscuous’’ pattern of recognition

and are characterized by a broad specificity for different HLA class I

molecules (LIR1/ILT2) or recognize the HLA-class Ib HLA-E molecules

(CD94/NKG2A)10–14.

This mechanism is sophisticated (KIRs detect allelic determinants

of HLA class I molecules) and of recent evolution since murine NK cells

lack KIRs (a similar function is mediated by structurally different

receptors10 and major differences in the type and specificity of expressed

KIRs exist in chimpanzees, a species that diverged from humans only

�

120

G.

FE

RL

AZ

ZO

5 million years ago15. This clearly means that KIRs have evolved recently,

paralleling the rapid evolution of HLA-class I molecules.

6.2 CYTOTOXIC NK CELLS AND CYTOKINE SECRETING NK CELLS

Although NK cells were initially described as cytotoxic effectors in peri-

pheral blood and were found to lyse tumor cells without prior activa-

tion, recent studies suggest that a possibly immunoregulatory subset of

NK cells responds to activation mainly with cytokine secretion. This

latter subset should play the prominent protective role during bacterial

infections. For instance, IFNg, a major cytokine released by activated

NK cells, represents the principal phagocyte-activating factor5, indicating

the crucial function of NK cell activation during bacterial infections.

Human peripheral blood mononuclear cells contain around 10% of

NK cells16. The majority (¸95%) belongs to the CD56dimCD16þ cytolytic

NK subset17–19. These cells carry homing markers for inflamed peripheral

sites and carry perforin to rapidly mediate cytotoxicity17,18. The minor NK

subset in blood (�5%) is CD56brightCD16� cells17–19. These NK cells lack

perforin (or have low levels of it), but secrete large amounts of IFNgand TNFb upon activation and are superior to CD56dim NK cells in these

functions18,19. In addition, they display homing markers for secondary

lymphoid organs, namely CCR7 and CD62L7.

Another difference between these subsets can be found in their

receptors mediating target recognition. Human NK cell recognition of

target cells is guided by the balance of activating and inhibitory signals given

by different groups of surface receptors. The main activating receptors

constitutively found on all NK cells in peripheral blood are NKG2D and the

nitrogen catabolite repressors (NCRs) NKp30 and NKp4620. All of them

probably recognize molecules that are upregulated upon cellular stress21,22.

However, only the stress-induced NKG2D ligands MICA/B and ULBPs have

so far been identified23,24. While cytotoxic peripheral blood CD56dim NK cells

are able to target antibody-opsonized cells via their low affinity FcgRIII/CD16

molecule, immunoregulatory CD56bright NK cells lack this receptor nearly

entirely18,19.

Most inhibitory NK cell receptors engage MHC class I molecules on

target cells12. They can be distinguished into two groups, detecting either

common allelic determinants of MHC class I, or MHC class I expression in

general. The KIR receptors constituting the first group distinguish

polymorphic HLA-A, -B and -C molecules. The inhibitory receptor surveying

MHC class I expression in general are more heterogeneous. They include the

�121

INT

ER

AC

TIO

NS

BE

TW

EE

NN

AT

UR

AL

KIL

LE

RA

ND

DE

ND

RIT

ICC

EL

LS

DU

RIN

GB

AC

TE

RIA

LIN

FE

CT

ION

S

LIR1/ILT2 molecule with a broad specificity for different HLA class I

molecules and the CD94/NKG2A heterodimer, specific for HLA-E whose

surface expression is dictated by the availability of HLA class I heavy chain

signal peptides. The MHC class I allele specific KIR receptors are expressed

on subsets of CD56dimCD16þ cytolytic NK cells, while the immunoregula-

tory CD56brightCD16� NK subset expresses uniformly CD94/NKG2A

and lacks KIRs18.

These phenotypes are consistent with the hypothesis that

CD56dimCD16þ NK cells are terminally differentiated effectors that carry

the whole panel of sophisticated activating and inhibitory receptors to even

detect allelic HLA loss and can readily lyse aberrant cells at peripheral

inflammation sites. On the contrary, CD56brightCD16� NK cells might

perform an immunoregulatory function in secondary lymphoid tissues

and release large amount of effector cytokines able to control pathogen

spreading.

6.3 NK CELLS IN BLOOD AND IN SECONDARY

LYMPHOID ORGANS

Peripheral blood is the most accessible source of human NK cells. Therefore,

most studies have been performed with NK cell populations from this organ

and it was assumed that most NK cells circulate in human blood after their

emigration from the bone marrow. Recently, however, it has been shown that

a substantial amount of human NK cells homes to secondary lymphoid

organs. These amount to around 5% of mononuclear cells in uninflamed

lymph nodes and 0.4–1% in inflamed tonsils and lymph nodes25,26. These

NK cells constitute a remarkable pool of innate effector cells, since lymph

nodes harbor 40% of all lymphocytes, while peripheral blood contains only

2% of all lymphocytes27,28. Therefore lymph node NK cells are in the absence

of infection and inflammation 10 times more abundant than blood NK cells.

As expected from CCR7 and CD62L expression on CD56bright NK cells in

blood, the CD56bright NK subset is enriched in all secondary lymphoid organs

analysed so far (lymph nodes, tonsils and spleen)26. In spleen, around 15%

and in tonsils and lymph nodes around 75% of NK cells belong to the

CD56bright subset. Lymph node and tonsil NK cells, however, uniformly lack

FcgRIII/CD16 and KIRs, while the CD56dim NK cells in spleen express these

molecules26. Surprisingly and in contrast to CD56bright blood NK cells, lymph

node and tonsil NK cells show no or very low expression of the constitutive

NCRs NKp30 and NKp4625,26. On the other hand, the inducible NCR

NKp44, undetectable on NK cells directly isolated from peripheral blood,

�

122

G.

FE

RL

AZ

ZO

is upregulated on NK cells harbored in inflamed tonsils26. Therefore, target

cell recognition by lymph node and tonsil NK cells seems to be mainly

influenced by NKG2D as activating and CD94/NKG2A as inhibitory

receptor25,26. This NK receptor repertoire of lymph node and tonsil NK

cells is similar to CD56bright blood NK cells, but even more restricted

by the absence of constitutive NCRs.

With respect to NK function, secondary lymphoid tissue NK cells show

an impressive plasticity. Although lymph node and tonsil NK cells are

initially perforin negative and show no cytolytic activity against MHC class I

low and ULBPhigh targets, perforin and cytotoxicity can be upregulated by

IL-2 within 3–7 days26. Interestingly, NK cells from secondary lymphoid

organs gain during the same time-period expression of CD16, NCRs NKp30,

NKp46 and NKp44 as well as KIRs26. Therefore, activation converts lymph

node and tonsil NK cells into effectors, similar to the terminally differ-

entiated CD56dimCD16þ blood NK cells with cytolytic function and the

sophisticated set of inhibitory and activating receptors.

6.4 DENDRITIC CELLS AS EARLY ACTIVATORS

OF NK CELL FUNCTIONS

The mentioned human NK cell compartments are probably all involved in

early innate immune responses. Recent studies have demonstrated that

during the innate phase of the immune response NK cells can also mediate

DC maturation29–31. This activation is not unidirectional, because the

interaction between mature, but not immature, DCs and NK cells results

in NK cell proliferation, IFNg production and induction of cytolytic

activity8,32,33. Thus, DCs have now emerged as the activators of NK response

in the early phases of the immune response, i.e. before an adaptive immune

response had been evoked and T cell derived cytokine, such as IL-2, could

be produced.

Remarkably, DC-induced NK cell cytolytic activity was directed not

only toward tumor cells, but also against immature DCs (iDCs). The

NK-mediated killing of DCs was mostly dependent on the NKp30 NCR.

Other activating receptors or co-receptors played virtually no role33. This

would imply that DCs express the ligand for NKp30, but not for other major

triggering NK receptors.

During NK activation by myeloid DCs both soluble factors as well as

cell-to-cell contact seem to be important.

In mice, induction of NK cell cytotoxicity was entirely blocked by trans-

well separation of DCs and NK cells, indicative for a major contribution of

�123

INT

ER

AC

TIO

NS

BE

TW

EE

NN

AT

UR

AL

KIL

LE

RA

ND

DE

ND

RIT

ICC

EL

LS

DU

RIN

GB

AC

TE

RIA

LIN

FE

CT

ION

S

DC surface receptors in NK activation32. In addition, DC derived IFNa/b, IL-

12 and IL-18 have been reported to be crucial in murine NK activation34–36.

While IL-12 was mainly implicated in NK mediated INFg secretion, IFNa/bseems required for cytotoxicity of NK cells35,37. In addition, secretion of

IL-2 by DCs stimulated with microbial stimuli might also contribute

to NK activation38,39.

In humans, NK activation by DCs was not significantly disrupted by

trans-well separation of the two cell types, indicating a major contribution of

soluble factors in NK activation40,41. In one study, DCs were unable to

activate NK cells in the presence of neutralizing antibodies for IL-12 and

IL-1841. Other studies demonstrated that NK activation by DC subsets

correlates with IL-12 secretion by these DCs, while IL-15 and IL-18 secretion

were not indicative for NK activation42,43. Therefore, IL-12 might play

an important role in human NK activation by DCs.

Apart from myeloid DCs that efficiently activate human and mouse

NK cells, plasmacytoid DCs (pDCs) might also contribute to NK activa-

tion. This DC subset has been found to produce 200 to 1000 times more

type I interferons than other blood cells after viral challenge44 and

IFNab are critical cytokines for inducing NK cell-mediated lysis of

virus-infected targets45–47. IFNab secretion seems, however, not only

the signature of pDCs, but also myeloid DCs can secrete substantial

amounts of these cytokines upon direct viral infection48, as well as

upon bacterial infection49. Therefore, the contributions of myeloid and

plasmacytoid DCs in type I interferon mediated NK activation remains

to be established.

While cytokines play a dominant role in human NK cell activation

by DCs, IFNa treatment of DCs in addition upregulates the NKG2D

ligands MICA/B on monocyte-derived DCs and these molecules seem to

activate resting NK cells in a cell contact dependent manner50. MICA/B

upregulation on DCs upon IFNa exposure seems to be mediated by

DC derived autocrine/paracrine IL-1551. Therefore, cell-contact might

contribute under certain inflammatory conditions to NK activation by

human DCs.

6.5 EFFECT OF BACTERIAL INFECTIONS ON

DC/NK CELL INTERACTIONS

It is conceivable that DC/NK cell interactions may occur primarily during

infections. Therefore it was important to analyze the effect of live bacteria

on the cross-talk between DCs and NK cells. Two different models

�

124

G.

FE

RL

AZ

ZO

of bacterial infection have been analyzed42. One represented by the

extracellular bacteria Escherichia coli (E. coli), the other by the intracellular

mycobacterium BCG, capable of efficiently infecting DCs52,53. In both

systems, bacterial infection of DCs led to a particularly rapid NK

cell activation.

6.5.1 Autologous DC-induced proliferation of NK cells is

enhanced by BCG

Since mature DCs are capable of inducing proliferation of autologous NK

cells, 33 it was then further investigated whether the infection of DCs with

BCG had any effect on this capability. BCG was employed as infective agent

because of its ability to efficiently infect DCs without undergoing substantial

proliferation (thus not interfering with 3H-thymidine incorporation assays).

In this study, it was confirmed that DCs, derived from monocytes in the

presence of GM-CSF and IL-4, were able to induce NK cells proliferation. It is

of note that both the NK cell proliferation and the number of viable NK cells

recovered after 5 days of culture were significantly increased in the presence

of BCG. Control experiments with culture containing NK cells and BCG

alone did not lead to NK cell proliferation.

The observed NK cell proliferation is likely to be sustained by

lymphokines such as IL-2 and IL-1538,54,55. Although these cytokines were

detected at extremely low levels in the supernatants derived from DCs

cultured in the presence of bacteria, it is not possible to exclude their role

in DC-mediated NK cell expansion. Indeed, a more recent study indicate

a relevant role of the membrane-bound form of IL-15 on human DCs

stimulated by different inflammatory stimuli, including LPS, in DC-

dependent NK cell proliferation. Notably, in this experimental model,

CD56bright lymph node NK cells were preferentially expanded during

DC/NK coculture8.

6.5.2 Effectiveness of BCG and E. coli in inducing an activated

NK cell phenotype

Previous studies have shown that, following stimulation with LPS, DCs

induce the expression, in NK cells, of the early activation marker CD6929,31.

Accordingly, DCs that had been exposed to living bacteria could also induce

activation markers on NK cells. Both CD69 and HLA-DR were also

upregulated in the presence of bacteria. It is of note that whereas CD69

and HLA-DR can be detected after interaction of NK cells with non-infected

�125

INT

ER

AC

TIO

NS

BE

TW

EE

NN

AT

UR

AL

KIL

LE

RA

ND

DE

ND

RIT

ICC

EL

LS

DU

RIN

GB

AC

TE

RIA

LIN

FE

CT

ION

S

immature DCs, DC infection resulted in a greater increase of the expression

of both molecules on NK cell surface. In addition, a de novo expression of

CD25 could be detected. This de novo expression may be functionally

relevant since the expression of CD25, a component of the IL-2 receptor

complex, renders NK cells highly responsive to IL-2. Neither BCG nor E. coli

could induce the expression of these activation markers in the absence

of DCs.

Since the NKp30 receptor is primarily involved in DCs recognition and

lysis by NK cells33, it was also analyzed whether mAb-mediated masking of

NKP30 could interfere with the NK cell activation induced by DCs and

bacteria. The addition of anti-NKp30 mAb did not modify the expression of

CD69, HLA-DR and CD25. These data clearly indicate that the induction of

an activated phenotype in NK cells is not mediated via NKp30. Therefore, this

triggering receptor, which plays a major role in the recognition and lysis of

DCs, does not appear to play any substantial role in the activating signal

delivered by DCs to NK cells.

6.5.3 DC editing by NK cells activated by DCs and bacteria

Activated NK cells can lyse autologous iDCs while they are less effective

against mature DCs33,56. In addition, a short term coculture of resting NK

cells with DCs that had been pulsed with LPS or heat-killed Mycobacterium

tuberculosis, has been reported to result in increase of NK-mediated

cytotoxicity against the Daudi target cell line29. It was further investigated

whether NK cells cocultured with DCs that had been infected with live

bacteria, could lyse autologous iDCs. After 24 h of coculture, only NK cells

cultured in the presence of infected DCs could lyse autologous iDCs. After

this time interval, uninfected iDCs failed to induce NK cell cytotoxicity.

Remarkably, infected DCs were less susceptible to the lysis, as compared

to iDCs cultured in the absence of bacteria. These results were obtained

after as few as 24 h of NK/DC coculture.

In the same set of experiments it was also analyzed whether polyclonal

NK cell lines cultured for over 1 week in IL-2 could discriminate between

infected and non-infected autologous DCs. IL-2-cultured NK cells lysed

uninfected iDCs very efficiently, but were less effective against infected DCs.

Therefore, the resistance of infected DCs to NK-mediated lysis does not

depend upon the degree of NK cell activation, but rather reflects an intrinsic

property of infected DCs themselves.

In this context, the arrival of NK cells to inflamed tissues and their

encounter with iDCs may appear paradoxical, as it would lead to depletion of

�

126

G.

FE

RL

AZ

ZO

antigen-presenting cells (APCs). Nevertheless, DCs exposed to bacteria

become highly resistant to NK cell-mediated lysis. Therefore, while they can

positively influence NK cells, they are not damaged by NK cells themselves

and are allowed to migrate to secondary lymphoid organs.

In turn, NK cells undergo both activation and proliferation, display

a rapid increase in their cytolytic activity and may release large amounts

of cytokines, including TNF-a, GM-CSF and IFNg54, which may further

amplify the inflammatory response.

6.5.4 The resistance of infected DCs to NK-mediated cytotoxicity

is due to the upregulation of HLA class I molecules

The different susceptibility of iDCs versus mature DCs is primarily related to

differences in surface expression of HLA class I molecules57. In order to

determine whether the early resistance of infected DCs to NK-mediated lysis

reflected a rapid upregulation of HLA class I molecules, the surface density

of HLA class I on DCs, cultured alone or in the presence of either BCG or E.

coli, was comparatively analyzed on a quantitative basis. Cell size and the

expression of HLA class I molecules were evaluated by flow cytometry. By the

simultaneous evaluation of cell size and fluorescence intensity the number

of HLA class I molecules/m2 of DCs cell surface could be calculated. Two days

(48 h) after infection, DCs increased the number of HLA class I molecules/m2

approximately ten-fold with both BCG and E. coli. In view of the high

expression of HLA class I in infected DCs, it appears conceivable that

resistance to NK-mediated lysis could be a distinct consequence of this

phenomenon. On the other hand, another possible explanation could be that

BCG and E. coli could down-regulate the expression of the ligands for

triggering receptors of NK cells. In order to discriminate between these two

possibilities, cytolytic tests in the presence of mAbs (IgM isotype) specific for

HLA class I molecules were performed. Upon mAb-mediated masking of

HLA class I molecules, a sharp increase of cytolytic activity against infected

DCs could be detected. This clearly indicates that the resistance of infected

DCs to NK-mediated lysis is due to increased inhibitory interactions

occurring between HLA class I and inhibitory receptors expressed on NK

cells. The anti-HLA class I mAb-induced restoration of the cytolytic activity

and the inhibition of this activity by anti-NKp30 mAb, manifestly pointed out

that infected DCs also express levels of ligands for this triggering NK receptor

sufficient to induce NK cell activation.

A relevant question related to the above-presented data is why

bacterial infection should lead to a rapid induction of NK cell activation

�127

INT

ER

AC

TIO

NS

BE

TW

EE

NN

AT

UR

AL

KIL

LE

RA

ND

DE

ND

RIT

ICC

EL

LS

DU

RIN

GB

AC

TE

RIA

LIN

FE

CT

ION

S

and cytotoxicity. Thus, while NK cell recruitment and activation result

in production of cytokines and chemokines which may contribute to the

defense against bacterial spreading, the cytolytic activity of NK cells does

not exert any direct effect against bacteria. In this regard, a physiopatho-

logic mechanism, in which activated NK cells could play a role in the

homeostasis of the immune response during bacterial infections, has been

proposed33,58. This model is based on the evidence that activated NK cells

are inefficient in killing infected DCs (as discussed above) but they can

efficiently lyse uninfected DCs. Accordingly, the presence of activated NK

cells in tissues and lymph nodes, inflamed because of bacterial infection,

may limit an overwhelming recruitment of iDCs at a stage in which bacteria

have already been eliminated and infection has been controlled. Therefore,

the ability of NK cells to discriminate between infected and uninfected

DCs may suggests that NK cells play an important regulatory role by

selectively editing APC during bacterial infection and thus switching off

an excessive immune response. This mechanism may be particularly useful

in preventing tissue damage.

Exposure of DCs to bacteria rapidly induce DC maturation and

expression of functionally important surface molecules, including CD80

and CD86 coreceptors, HLA molecules and CCR742. Thus, DCs acquire

rapidly the ability to efficiently function as professional APC and to migrate

to secondary lymphoid organs, where they can interact with Tcells and evoke

a prompt adaptive immune response against infecting bacteria. In addition,

DCs that had encountered bacteria can also rapidly and markedly potentiate

an important effector arm of the innate immunity by inducing a rapid

activation of NK cells.

A conceivable question may now be how and where DCs and NK cells

can meet each other.

6.6 SITES OF NK/DC INTERACTION DURING

BACTERIAL INFECTION

6.6.1 DC/NK cell cross-talk at sites of bacterial invasion

The recent data on NK cell activation by DCs suggest that these APCs initiate

the early and innate NK activation during the immune response. The

question remains as to where this interaction takes place. No direct evidence

exists that DCs and NK cells encounter at sites of bacterial infection.

Nevertheless, microbial invasion causes tissue inflammation and one site of

DC/NK interaction is inflamed tissue. NK cells have been found in close

contact to DCs in lesions of allergen induced atopic eczema/dermatitis

�

128

G.

FE

RL

AZ

ZO

syndrome59. Moreover, the chemokine receptor repertoire and the chemo-

kine responsiveness of CD56dimCD16þ blood NK cells suggest that they can

home to sites of inflammation efficiently17. The cytotoxic blood NK subset

migrates efficiently in response to IL-8 and soluble fractalkine and expresses

the respective receptors for these chemokines, CXCR1 and CX3CR1. Both

chemokines are induced by proinflammatory cytokines like IL-1 and

TNFa60,61. Fractalkine mediates adhesion to endothelia and emigration of

NK cells from the blood stream, while IL-8 mediates further migration to

the site of inflammation62. Therefore, cytotoxic CD56dimCD16þ blood

NK cells are able to home to inflamed tissues where they can encounter

dendritic cells, which are resident in peripheral tissue sites.

The DC/NK encounter at sites of inflammation can either result in DC

maturation by modest NK infiltration or in immature DC lysis due to large

NK cell infiltrates31. The maturation of DCs upon NK encounter has been

largely attributed to TNFa secretion by NK cells29,31. Both NK effector

mechanisms will deplete the inflamed tissue of DCs either by maturation-

induced migration or by killing. This will deprive DC trophic bacteria of their

host cells at the site of infection.

6.6.2 DCs infected by bacteria activate and induce maturation

of NK cells in secondary lymphoid organs

Until recently only limited information had been available on NK cells

located in lymphoid tissues, and therefore NK cells have mainly been

considered as effector cells harbored in the blood stream and able to

promptly extravasate to inflamed tissues. The evidence that a large

amount of NK cells is located in uninflamed lymph nodes26 suggests

secondary lymphoid organs as important sites of NK cell activation.

Indeed, CD56brightCD16� NK cells isolated from uninflamed human

lymph nodes become strongly cytolytic upon stimulation with IL-2.

The de novo acquired cytotoxic properties were accompanied by the

expression of both activating and inhibitory receptors.

In addition, perforin negative NK cells located in secondary

lymphoid organs might play different roles prior to maturation into cyto-

lytic effectors, such as secretion of critical immunoregulatory cytokines

upon activation. In this regard, it has been demonstrated that peripheral

blood CD56brightCD16� NK cells produce significantly higher levels

of cytokines than their CD56dimCD16þ counterpart19,54. Similarly, the

CD56brightCD16� NK subset located in secondary lymphoid organs pro-

duces relevant cytokines prior to maturation into cytolytic effector cells26.

�129

INT

ER

AC

TIO

NS

BE

TW

EE

NN

AT

UR

AL

KIL

LE

RA

ND

DE

ND

RIT

ICC

EL

LS

DU

RIN

GB

AC

TE

RIA

LIN

FE

CT

ION

S

Interestingly, several reports have recently shown that DCs elicit

IFNg secretion by autologous NK cells29–33. Specifically, NK cells from

secondary lymphoid organs are particularly effective in this function and

when they are cocultured with autologous DCs stimulated by bacterial

products can produce IFNg within 6 h8. This is of interest since IFNgrepresents a main cytokine for phagocyte activation. Indeed, CD56bright

CD16� NK cells produce significantly higher levels of IFNg, TNFb,

GM-CSF, IL-10 and IL-13 protein in response to monokines produced

by DCs, such as IL-12, IL-15, IL-18 and IL-1b19. Based on these findings, NK

cells in secondary lymphoid organs should not be referred to as merely

‘‘immature’’ NK cells but rather as effector cells whose functional plasticity

enables them to accomplish different sequential tasks during immune

responses.

Since DCs mature and migrate to secondary lymphoid tissues

following an encounter with bacteria, they might encounter CD56bright

CD16� NK cells there in the very early phase of an immune response

prior to T cell activation. As discussed in detail below, this could result in

local cytokine release by NK cells, which might be able to shape the follow-

ing adaptive immune response and probably also APC functions63.

6.7 ROLES FOR NK CELLS IN DC-MEDIATED

T CELL POLARIZATION

We discussed above that a recent ‘‘in vitro’’ model proposed that NK cells

might play an important regulatory role by selectively editing APC during

the course of immune responses. NK-mediated lysis of immature, but

not mature DCs might select an immunogenic DC population during the

initiation of immune responses.

In addition to removal of non-immunogenic DCs, NK cells also

secrete IFNg upon encounter with DCs33. As a consequence, subsequent

T cell polarization may be influenced. Indeed, in vitro studies in both

mouse and human systems have demonstrated the importance of IFNgin the polarization of type 1 immune response. Interestingly, in a murine

model of skin graft rejection, the recognition of donor DCs by host NK cells

led to modulation of Th1/Th2 cell development. Namely, turning host

NK cells off was sufficient to skew the alloresponse to Th264.

Thus, as already mentioned, the encounter of mature DCs with

perforinneg NK cells located in secondary lymphoid organs should lead to

a critical immunoregulatory role65, as DCs can induce NK cells of human

secondary lymphoid organs to secrete IFNg8. Moreover, DCs selectively

�

130

G.

FE

RL

AZ

ZO

Fig

ure

6.1

.D

uri

ng

bac

teri

alin

fect

ion

DC

san

dN

Kce

lls

can

inte

ract

inti

ssu

esin

flam

edb

yb

acte

rial

inva

sio

nan

din

seco

nd

ary

lym

ph

oid

org

ans.

Up

on

bac

teri

al

infe

ctio

n,

NK

cell

sg

etre

cru

ited

fro

mp

erip

her

alb

loo

dto

infl

amed

tiss

ues

(up

per

left

).C

yto

lyti

c,p

erfo

rinþ

CD56

dim

NK

cell

s,w

hic

hco

nst

itu

teth

em

ajo

rity

of

per

iph

eral

blo

od

NK

cell

s,ca

rry

the

nec

essa

rych

emo

kin

ere

cep

tors

toh

om

eto

site

so

fin

flam

mat

ion

.Th

eyca

ned

itD

Cs

her

eb

efo

reth

ese

cell

sm

atu

rean

dm

igra

teto

seco

nd

ary

lym

ph

oid

org

ans.

Inad

dit

ion

,u

po

nen

cou

nte

rw

ith

DC

s,N

Kce

lls

can

rele

ase

cyto

kin

esth

atca

nco

ntr

ol

bac

teri

alsp

read

ing

bu

tal

soin

du

ceo

rm

od

ula

te

infl

amm

ato

ryre

spo

nse

s,as

wel

las

con

tro

lth

eg

row

than

dfu

nct

ion

of

mo

no

cyte

s/m

acro

ph

ages

,g

ran

ulo

cyte

san

dD

Cs

them

selv

es.

Inse

con

dar

yly

mp

ho

ido

rgan

s

lik

ely

mp

hn

od

es,

DC

sca

nm

eet

wit

hre

gu

lato

ryC

D56

bri

gh

tN

Kce

lls

(lo

wer

rig

ht)

,w

hic

hco

nst

itu

teth

em

ajo

rity

of

NK

cell

sin

seco

nd

ary

lym

ph

oid

tiss

ues

.T

he

inte

ract

ion

bet

wee

nD

Cs

mat

ure

db

yb

acte

ria

and

CD56

bri

gh

tN

Kce

lls

ensu

rea

larg

ere

leas

eo

fIF

Ng

by

NK

cell

svi

aD

C-d

eriv

edL

-12

.Th

ere

alea

seo

fIF

Ng

and

/or

oth

er

imm

un

om

od

ula

tory

cyto

kin

esin

flu

ence

the

mag

nit

ud

ean

dth

ep

ola

riza

tio

no

fth

eD

C-i

nit

iate

dad

apti

veim

mu

ne

resp

on

se.

stimulate the CD56brightCD16neg cell subset to produce IFNg and the

production is extremely rapid and fully dependent on IL-12 released by

mature DCs8. In vivo, murine lymph node NK cells, activated by LPS-

stimulated autologous DCs migrating into the lymph node, secrete IFNgwith a peak of cytokine release after 48 h. NK cell depletion and

reconstitution experiments show that NK cells provide an early source

of IFNg that is absolutely required for Th1 polarization9. Therefore, in

a model of DC stimulation by bacterial products, NK cells play a crucial

regulatory role during DC-dependent T cell priming and subsequent

polarization in the T cell areas of secondary lymphoid organs.

In conclusion, early activation of NK cells by DCs activated by

bacteria may play an immunoregulatory role in shaping the emerging

adaptive immune responses. The ability to edit APC as well as secrete

immunomodulatory cytokines might result in increased and predominantly

Th1 polarized immune responses.

In summary, immunomodulatory as well as cytotoxic NK cells can

be activated via dendritic cells infected by bacteria early during the

immune responses. Possible interaction sites for this encounter are

sites of inflammation and secondary lymphoid tissues (Figure 6.1).

DC-activated NK cells might then exert effector functions against infected

cells, but mainly influence the emerging adaptive immune response via

APC editing and immunomodulatory cytokine secretion.

The complex cross talk occurring between these two major players

of the innate immunity provides a novel mechanism by which NK cells

could cooperate in the defense against bacteria.

REFERENCES

1. Trinchieri, G. (1989). Biology of natural killer cells. Adv. Immunol.

7, 176–87.

2. Mingari, M. C., A. Poggi, R. Biassoni, R. Bellomo, E. Ciccone, N. Pella,

et al. (1991). In vitro proliferation and cloning of CD3�CD16þ cells

from human thymocyte precursors. J. Exp. Med. 174, 21–6.

3. Rodewald, H. R., P. Moingeon, J. L. Lucich, C. Dosiou, P. Lopez, and

E. L. Reinherz (1992). A population of early fetal thymocytes expressing

FcgRII/III contains precursors of T lymphocytes and natural killer

cells. Cell 69, 139–49.

4. Lanier, L. L., H. Spits, and J. H. Phillips (1992). The developmental

relationship between NK cells and T cells. Immunol. Today 13, 392–9.

�

132

G.

FE

RL

AZ

ZO

5. Boehm, U., T. Klamp, M. Groot, and J. C. Howard (1997). Cellular

responses to interferon-gamma. Annu. Rev. Immunol. 15, 749–95.

6. Mingari, M. C., C. Vitale, C. Cantoni, R. Bellomo, M. Ponte, F. Schiavetti,

et al. (1997). Interleukin-15-induced maturation of human natural

killer cells from early thymic precursors. Selective expression of

CD94/NKG2A as the only HLA-class I specific inhibitory receptor.

Eur. J. Immunol. 27, 1374–80.

7. Ljunggren, H. G. and K. Karre (1990). In search of the ‘‘missing self’’.

MHC molecules and NK cell recognition. Immunol. Today 11, 237–44.

8. Ferlazzo, G., M. Pack, D. Thomas, C. Paludan, D. Schmid, T. Strowig,

G. Bougras, W. A. Muller, L. Moretta, and C. Munz (2004). Distinct

roles of IL-12 and IL-15 in human natural killer cell activation by

dendritic cells from secondary lymphoid organs. Proc. Natl Acad. Sci. U S A

101, 16606–11.

9. Martin-Fontecha, A., L. L. Thomsen, S. Brett, C. Gerard, M. Lipp, A.,

Lanzavecchia, and F. Sallusto (2004). Induced recruitment of NK cells

to lymph nodes provides IFN-gamma for T(H)1 priming. Nat. Immunol.

5, 1260–5.

10. Yokoyama, W. M. and W. E. Seaman (1993). The Ly49 and NKR-P1

gene families encoding lectin-like receptors on natural killer cells: the

NK gene complex. Annu. Rev. Immunol. 11, 613–35.

11. Moretta, A., C. Bottino, M. Vitale, et al. (1996). Receptors for

HLA-class I-molecules in human natural killer cells. Annu. Rev. Immunol.

14, 619–48.

12. Lanier L. L. (1998). NK cell receptors. Annu. Rev. Immunol. 16, 359–93.

13. Long, E. O. (1999). Regulation of immune response through inhibitory

receptors. Annu. Rev. Immunol. 17, 875–904.

14. Lopez-Botet, M., M. Perez Villar, M. Carretero, et al. (1997). Structure

and function of the CD94 C-type lectin receptor complex involved in the

recognition of HLA class I molecules. Immunol. Rev. 155, 165–74.

15. Khakoo, S. I., R. Rajalingam, B. P. Shum, et al. (2000). Rapid evolution

of NK cell receptor systems demonstrated by comparison of chimpanzees

and humans. Immunity 12, 687–98.

16. Robertson, M. J. and J. Ritz (1990). Biology and clinical relevance of

human natural killer cells. Blood 76, 2421–38.

17. Campbell, J. J., S. Qin, D. Unutmaz, D. Soler, K. E. Murphy,

M. R. Hodge, L. Wu, and E. C. Butcher (2001). Unique subpopulations

of CD56þ NK and NK-T peripheral blood lymphocytes identified

by chemokine receptor expression repertoire. J. Immunol. 166,

6477–82.

�133

INT

ER

AC

TIO

NS

BE

TW

EE

NN

AT

UR

AL

KIL

LE

RA

ND

DE

ND

RIT

ICC

EL

LS

DU

RIN

GB

AC

TE

RIA

LIN

FE

CT

ION

S

18. Jacobs, R., G. Hintzen, A. Kemper, K. Beul, S. Kempf, G. Behrens,

K. W. Sykora, and R. E. Schmidt (2001). CD56bright cells differ in their

KIR repertoire and cytotoxic features from CD56dim NK cells. Eur. J.

Immunol. 31, 3121–7.

19. Cooper, M. A., T. A. Fehniger, S. C. Turner, K. S. Chen, B. A. Ghaheri,

T. Ghayur, W. E. Carson, and M. A. Caligiuri (2001). Human natural

killer cells: a unique innate immunoregulatory role for the CD56bright

subset. Blood 97, 3146–51.

20. Moretta, A., C. Bottino, M. Vitale, D. Pende, C. Cantoni, M. C. Mingari,

R. Biassoni, and L. Moretta (2001). Activating receptors and coreceptors

involved in human natural killer cell-mediated cytolysis. Annu. Rev.

Immunol. 19, 197–223.

21. Moretta, L., C. Bottino, D. Pende, M. C. Mingari, R. Biassoni, and

A. Moretta (2002). Human natural killer cells: their origin, receptors

and function. Eur. J. Immunol. 32, 1205–11.

22. Moretta, L., G. Ferlazzo, M. C. Mingari, G. Melioli, and A. Moretta (2003).

Human natural killer cell function and their interactions with dendritic

cells. Vaccine 21, Suppl. 2, S38.

23. Bauer, S., V. Groh, J. Wu, A. Steinle, J. H. Phillips, L. L. Lanier, and

T. Spies (1999). Activation of NK cells and T cells by NKG2D, a receptor

for stress- inducible MICA. Science 285, 727–9.

24. Cosman, D., J. Mullberg, C. L. Sutherland, W. Chin, R. Armitage,

W. Fanslow, M. Kubin, and N. J. Chalupny (2001). ULBPs, novel MHC

class I-related molecules, bind to CMV glycoprotein UL16 and

stimulate NK cytotoxicity through the NKG2D receptor. Immunity 14,

123–33.

25. Fehniger, T. A., M. A. Cooper, G. J. Nuovo, M. Cella, F. Facchetti,

M. Colonna, and M. A. Caligiuri (2003). CD56bright natural killer cells

are present in human lymph nodes and are activated by T cell-derived

IL-2: a potential new link between adaptive and innate immunity.

Blood 101, 3052–7.

26. Ferlazzo, G., S. L. Lin, K. Goodman, D. Thomas, B. Morandi, W. A. Muller,

A. Moretta, and C. Munz (2004). The abundant NK cells in human

lymphoid tissues require activation to become cytolytic. J. Immunol.

172, 1455–62.

27. Westermann, J. and R. Pabst (1992). Distribution of lymphocyte

subsets and natural killer cells in the human body. Clin. Investig. 70,

539–44.

28. Trepel, F. (1974). Number and distribution of lymphocytes in man.

A critical analysis. Klin. Wochenschr. 52, 511–15.

�

134

G.

FE

RL

AZ

ZO

29. Gerosa, F., B. Baldani-Guerra, C. Nisii, V. Marchesini, G. Carra, and

G. Trinchieri (2002). Reciprocal activating interaction between natural

killer cells and dendritic cells. J. Exp. Med. 195, 327–33.

30. Gerosa, F., A. Gobbi, P. Zorzi, S. Burg, F. Briere, G. Carra, and G. Trinchieri

(2005). The reciprocal interaction of NK cells with plasmacytoid or

myeloid dendritic cells profoundly affects innate resistance functions.

J. Immunol. 174, 727–34.

31. Piccioli, D., S. Sbrana, E. Melandri, and N. M. Valiante (2002).

Contact-dependent stimulation and inhibition of dendritic cells by

natural killer cells. J. Exp. Med. 195, 335–41.

32. Fernandez, N. C., A. Lozier, C. Flament, P. Ricciardi-Castagnoli,

D. Bellet, M. Suter, M. Perricaudet, T. Tursz, E. Maraskovsky, and

L. Zitvogel (1999). Dendritic cells directly trigger NK cell functions:

cross-talk relevant in innate anti-tumor immune responses in vivo.

Nat. Med. 5, 405–11.

33. Ferlazzo, G., M. L. Tsang, L. Moretta, G. Melioli, R. M. Steinman, and

C. Munz (2002). Human dendritic cells activate resting natural killer

(NK) cells and are recognized via the NKp30 receptor by activated NK cells.

J. Exp. Med. 195, 343–51.

34. Andrews, D. M., A. A. Scalzo, W. M. Yokoyama, M. J. Smyth, and

M. A. Degli-Esposti (2003). Functional interactions between

dendritic cells and NK cells during viral infection. Nat. Immunol. 4, 175–81.

35. Dalod, M., T. Hamilton, R. Salomon, T. P. Salazar-Mather, S. C. Henry,

J. D. Hamilton, and C. A. Biron (2003). Dendritic cell responses to

early murine cytomegalovirus infection: subset functional specialization

and differential regulation by interferon alpha/beta. J. Exp. Med.

197, 885–98.

36. Orange, J. S. and C. A. Biron (1996). An absolute and restricted

requirement for IL-12 in natural killer cell IFN-g production and

antiviral defense. Studies of natural killer and T cell responses in

contrasting viral infections. J. Immunol. 156, 1138–42.

37. Nguyen, K. B., T. P. Salazar-Mather, M. Y. Dalod, J. B. Van Deusen,

X. Q. Wei, F. Y. Liew, M. A. Caligiuri, J. E. Durbin, and C. A. Biron (2002).

Coordinated and distinct roles for IFN-alpha beta, IL-12, and

IL-15 regulation of NK cell responses to viral infection. J. Immunol.

169, 4279–87.

38. Granucci, F., C. Vizzardelli, N. Pavelka, S. Feau, M. Persico, E. Virzi,

M. Rescigno, G. Moro, and P. Ricciardi-Castagnoli (2001). Inducible IL-2

production by dendritic cells revealed by global gene expression analysis.

Nat. Immunol. 2, 882–8.

�135

INT

ER

AC

TIO

NS

BE

TW

EE

NN

AT

UR

AL

KIL

LE

RA

ND

DE

ND

RIT

ICC

EL

LS

DU

RIN

GB

AC

TE

RIA

LIN

FE

CT

ION

S

39. Granucci, F., S. Feau, V. Angeli, F. Trottein, and P. Ricciardi-Castagnoli

(2003). Early IL-2 production by mouse dendritic cells is the result of

microbial-induced priming. J. Immunol. 170, 5075–81.

40. Nishioka, Y., N. Nishimura, Y. Suzuki, and S. Sone (2001). Human

monocyte-derived and CD83þ blood dendritic cells enhance NK

cell-mediated cytotoxicity. Eur. J. Immunol. 31, 2633–41.

41. Yu, Y., M. Hagihara, K. Ando, B. Gansuvd, H. Matsuzawa, T. Tsuchiya,

Y. Ueda, H. Inoue, T. Hotta, and S. Kato (2001). Enhancement of human

cord blood CD34þ cell-derived NK cell cytotoxicity by dendritic cells.

J. Immunol. 166, 1590–1600.

42. Ferlazzo, G., B. Morandi, A. D’Agostino, R. Meazza, G. Melioli, A. Moretta,

and L. Moretta (2003). The interaction between NK cells and dendritic

cells in bacterial infections results in rapid induction of NK cell activation

and in the lysis of uninfected dendritic cells. Eur. J. Immunol. 33, 306–13.

43. Munz, C., T. Dao, G. Ferlazzo, M. A. de Cos, K. Goodman, and J. W. Young

(2005). Mature myeloid dendritic cell subsets have distinct roles for

activation and viability of circulating human natural killer cells. Blood

105, 266–73.

44. Siegal, F. P., N. Kadowaki, M. Shodell, P. A. Fitzgerald-Bocarsly, K. Shah,

S. Ho, S. Antonenko, and Y. J. Liu (1999). The nature of the principal type 1

interferon-producing cells in human blood. Science 284, 1835–7.

45. Bandyopadhyay, S., B. Perussia, G. Trinchieri, D. S. Miller, and S. E. Starr

(1986). Requirement for HLA-DRþ accessory cells in natural killing of

cytomegalovirus-infected fibroblasts. J. Exp. Med. 164, 180–95.

46. Dalod, M., T. P. Salazar-Mather, L. Malmgaard, C. Lewis, C. Asselin-Paturel,

F. Briere, G. Trinchieri, and C. A. Biron (2002). Interferon a/b and

interleukin 12 responses to viral infections: pathways regulating dendritic

cell cytokine expression in vivo. J. Exp. Med. 195, 517–28.

47. Feldman, M., D. Howell, and P. Fitzgerald-Bocarsly (1992). Interferon-

a-dependent and -independent participation of accessory cells in natural

killer cell-mediated lysis of HSV-1-infected fibroblasts. J. Leukoc. Biol.

52, 473–82.

48. Diebold, S. S., M. Montoya, H. Unger, L. Alexopoulou, P. Roy, L. E. Haswell,

A. Al-Shamkhani, R. Flavell, P. Borrow, and C. Reis e Sousa (2003). Viral

infection switches non-plasmacytoid dendritic cells into high interferon

producers. Nature 424, 324–8.

49. Granucci, F., I. Zanoni, N. Pavelka, S. L. Van Dommelen, C. E. Andoniou,

F. Belardelli, M. A. Degli Esposti, and P. Ricciardi-Castagnoli (2004).

A contribution of mouse dendritic cell-derived IL-2 for NK cell activation.

J. Exp. Med. 200, 287–95.

�

136

G.

FE

RL

AZ

ZO

50. Jinushi, M., T. Takehara, T. Kanto, T. Tatsumi, V. Groh, T. Spies, T. Miyagi,

T. Suzuki, Y. Sasaki, and N. Hayashi (2003). Critical role of MHC

class I-related chain A and B expression on IFN-a-stimulated dendritic cells

in NK cell activation: impairment in chronic hepatitis C virus infection.

J. Immunol. 170, 1249–56.

51. Jinushi, M., T. Takehara, T. Tatsumi, T. Kanto, V. Groh, T. Spies, T. Suzuki,

T. Miyagi, and N. Hayashi (2003). Autocrine/paracrine IL-15 that is

required for type I IFN-mediated dendritic cell expression of MHC

class I-related chain A and B is impaired in hepatitis C virus infection.

J. Immunol. 171, 5423–9.

52. Inaba, K., M. Inaba, M. Naito, and R. M. Steinman (1993). Dendritic cell

progenitors phagocytose particulates, including bacillus Calmette-Guerin

organisms, and sensitize mice to mycobacterial antigens in vivo. J. Exp.

Med. 178, 479–88.

53. Demangel, C., A. G. Bean, E. Martin, C. G. Feng, A. T. Kamath, and

W. J. Britton (1999). Protection against aerosol Mycobacterium tuberculosis

infection using Mycobacterium bovis Bacillus Calmette Guerin-infected

dendritic cells. Eur. J. Immunol. 29, 1972–9.

54. Cooper, M. A., T. A. Fehniger, and M. A. Caligiuri (2001). The biology of

human natural killer-cell subsets. Trends Immunol. 22, 633–40.

55. Mattei, F., G. Schiavoni, F. Belardelli, and D. F. Tough (2001). IL-15

is expressed by dendritic cells in response to type I IFN, double-stranded

RNA, or lipopolysaccharide and promotes dendritic cell activation.

J. Immunol. 167, 1179–87.

56. Wilson, J. L., L. C. Heffler, J. Charo, A. Scheynius, M. T. Bejarano, and

H. G. Ljunggren (1999). Targeting of human dendritic cells by autologous

NK cells. J. Immunol. 163, 6365–70.

57. Ferlazzo, G., C. Semino, and G. Melioli (2001). HLA class I molecule

expression is up-regulated during maturation of dendritic cells, protecting

them from natural killer cell-mediated lysis. Immunol. Lett. 76, 37–41.

58. Moretta, A. (2002). Natural killer and dendritic cells: rendezvous in abused

tissues. Nat. Rev. Immunol. 2, 1–8.

59. Buentke, E., L. C. Heffler, J. L. Wilson, R. P. Wallin, C. Lofman,

B. J. Chambers, H. G. Ljunggren, and A. Scheynius (2002). Natural

killer and dendritic cell contact in lesional atopic dermatitis

skin-Malassezia-influenced cell interaction. J. Invest. Dermatol.

119, 850–7.

60. Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi,

D. R. Greaves, A. Zlotnik, and T. J. Schall (1997). A new class of

membrane-bound chemokine with a CX3C motif. Nature 385, 640–4.

�137

INT

ER

AC

TIO

NS

BE

TW

EE

NN

AT

UR

AL

KIL

LE

RA

ND

DE

ND

RIT

ICC

EL

LS

DU

RIN

GB

AC

TE

RIA

LIN

FE

CT

ION

S

61. Hoffmann, E., O. Dittrich-Breiholz, H. Holtmann, and M. Kracht (2002).

Multiple control of interleukin-8 gene expression. J. Leukoc. Biol.

72, 847–55.

62. Nishimura, M., H. Umehara, T. Nakayama, O. Yoneda, K. Hieshima,

M. Kakizaki, N. Dohmae, O. Yoshie, and T. Imai (2002). Dual functions

of fractalkine/CX3C ligand 1 in trafficking of perforinþ/granzyme Bþ

cytotoxic effector lymphocytes that are defined by CX3CR1 expression.

J. Immunol. 168, 6173–80.

63. Dowdell, K. C., D. J. Cua, E. Kirkman, and S. A. Stohlman (2003).

NK cells regulate CD4 responses prior to antigen encounter. J. Immunol.

171, 234–9.

64. Coudert, J. D., C. Coureau, and J. C. Guery (2002). Preventing NK cell

activation by donor dendritic cells enhances allospecific CD4 T cell

priming and promotes Th type 2 responses to transplantation antigens.

J. Immunol. 169, 2979–87.

65. Ferlazzo, G. and C. Munz (2004). NK cell compartments and their

activation by dendritic cells. J. Immunol. 172, 1333–9.

�

138

G.

FE

RL

AZ

ZO

PART III Dendritic cells and adaptiveimmune responses to bacteria

CHAPTER 7

Peculiar ability of dendritic cells to processand present antigens from vacuolarpathogens: a lesson from Legionella

Sunny Shin, Catarina Nogueira and Craig R. RoyYale University School of Medicine

7.1 L. PNEUMOPHILA AND LEGIONNAIRES’ DISEASE

Legionella pneumophila is a Gram-negative facultative intracellular patho-

gen capable of growing in both protozoan and mammalian host cells.

L. pneumophila is found in natural and artificial water reservoirs

and less often in soil and organic matter (Fields, 1996; Szymanska et al.,

2004). Optimal proliferation conditions for Legionella are those in which

water temperatures are between 25�C and 42�C, calcium and magnesium

salt-containing sediments are present, and are further enhanced by the

presence of algae and protozoa (Szymanska et al., 2004). In hostile

conditions, Legionella and other organisms become attached to surfaces

in an aquatic environment, forming a biofilm (Langmark et al., 2005).

L. pneumophila can be isolated from such natural water sources as

lakes, ponds and streams; however, artificial reservoirs such as plumbing

fixtures, hot water tanks, whirlpool spas and cooling towers, all possess

excellent conditions for Legionella proliferation inside protozoan hosts

and are the source of most outbreaks (Fliermans et al., 1981; Yee and

Wadowsky, 1982).

The first recognized outbreak of L. pneumophila occurred in

Philadelphia in 1976 during a state convention of the American Legion

(Fraser et al., 1977). During this outbreak a total of 221 people contracted the

disease, 34 of whom subsequently died. A new Gram-negative bacterium

was isolated from both patients and the air-conditioning system of the

hotel that was the source of the outbreak (McDade et al., 1977). This

isolated organism was named Legionella pneumophila (Brenner et al., 1979).

There are 48 different species of Legionella found in nature. Interestingly,

Legionella pneumophila serogroup 1, the strain isolated in the Philadelphia

outbreak, is responsible for most community-acquired outbreaks of

Legionnaires’ disease, followed by serogroups 4 and 6.

Legionella is acquired by inhalation of contaminated aerosolized water

droplets, resulting in bacterial entry into the human lung. Bacteria that

escape clearance by the action of lung cilia are ingested by alveolar

macrophages. Instead of being killed by alveolar macrophages, Legionella

establishes a vacuole that supports intracellular growth similar to that

observed in protozoan phagocytes (Horwitz, 1983). Infection with

L. pneumophila causes legionellosis, which comprises three possible

distinct clinical outcomes: (1) an atypical severe pneumonia described as

Legionnaires’ disease; (2) a flu-like form having a mild course known

as Pontiac fever; (3) an extra-pulmonary form in immunosuppressed

patients, often taking a severe clinical course, with septic syndrome,

coagulation disorders, acute cardiovascular deficiency and nephritis

(Szymanska et al., 2004). People of any age can acquire Legionnaires’

disease but the illness more often affects middle-aged and older patients,

particularly those who smoke cigarettes, and individuals who have

chronic respiratory diseases or renal deficiency. Also at increased risk are

individuals immunocompromised by diseases such as cancer, kidney

failure requiring dialysis, or diabetes. As the use of devices capable of

aerosolizing contaminated water have become more common, epidemics

of pneumonia that involve Legionella have also become more prevalent.

Those that are exposed to water aerosols (workers of cooling towers,

turbine operators and others) are at higher risk (Szymanska et al., 2004).

7.2 LEGIONELLA REPLICATES IN A SPECIALIZED VACUOLE

DERIVED FROM THE HOST ENDOPLASMIC RETICULUM

After internalization by phagocytes, L. pneumophila evades transport

to a lysosomal compartment, and establishes a replicative niche distinct

from the normal endocytic pathway (Roy and Tilney, 2002). The replicative

vacuole established by L. pneumophila is remodeled by early secretory

vesicles traveling between the host endoplasmic reticulum (ER) and Golgi

apparatus. The mature vacuole in which L. pneumophila replicates is

decorated with ribosomes and is similar to the host ER (Tilney et al., 2001).

The ability of L. pneumophila to alter trafficking of the vacuole in which it

resides is dependent upon a bacterial secretion apparatus called the Dot/Icm

system (Segal et al., 1998; Vogel et al., 1998). Upon encountering host cells,

the Dot/Icm system transfers bacterial proteins directly into the host cytosol

(Nagai et al., 2002; Chen et al., 2004; Luo and Isberg, 2004). These bacterial

�

142

S.

SH

IN,

C.

NO

GU

EIR

AA

ND

C.R

.R

OY

proteins engage host factors involved in vesicular transport and redirect the

vacuole containing L. pneumophila to prevent endocytic maturation and

promote remodeling by ER-derived vesicles (Horwitz, 1983; Tilney et al.,

2001; Kagan and Roy, 2002). Intracellular growth mutants of L. pneumophila

that are defective in the Dot/Icm system are unable to alter transport of their

vacuole and reside in vacuoles that mature similarly to phagosomes contain-

ing inert particles or non-pathogenic bacteria (Horwitz, 1987; Marra et al.,

1992).

7.3 THE BIRC1E PROTEIN MEDIATES HOST RESISTANCE

TO LEGIONELLA

Primary macrophages isolated from humans, guinea pigs, and hamsters

permit the intracellular replication of L. pneumophila, whereas, macrophages

isolated from most mouse strains do not allow for L. pneumophila growth.

Interestingly, macrophages from A/J mice are permissive for L. pneumophila

growth (Yamamoto et al., 1988). This phenotype translates to the in vivo

setting, with A/J mice capable of supporting L. pneumophila infection in

a manner comparable to the human course of disease (Brieland et al.,

1994). The susceptibility of A/J mice to L. pneumophila infection is con-

trolled by a single genetic locus called Lgn1. Birc1e (also called Naip5)

was positionally cloned as the gene within this locus responsible for

the permissive phenotype (Diez et al., 2003; Wright et al., 2003).

Genetic polymorphisms in Birc1e thus appear to control the ability of

L. pneumophila to replicate in mouse macrophages (Diez et al., 2003;

Wright et al., 2003). Birc1e is a member of the recently discovered

Nod-LRR (nucleotide oligomerization domain-leucine-rich repeat) family

of cytosolic pathogen recognition receptors (PRRs) (Inohara et al., 2004; Ting

and Williams, 2005). Nod-LRRs are thought to be responsible for dealing

with intracellular pathogens that might evade detection by Toll-like receptors

(TLRs). The Nod proteins have some homology to TLRs in that they contain

LRR domains thought to be responsible for detection of bacterial products

(Inohara et al., 2004; Ting and Williams, 2005). Birc1e is predicted to contain

three baculoviral inhibitors of apoptosis repeat (BIR) domains, a NOD

region and an LRR domain (Inohara et al., 2004; Ting and Williams, 2005).

The presence of BIR domains makes Birc1e similar to proteins in the

inhibitors of apoptosis (IAP) family. The molecular mechanism by which

Birc1e controls host resistance to L. pneumophila is currently unknown. A

detailed analysis of the fate of L. pneumophila in infected C57Bl/6

�143

PE

CU

LIA

RA

BIL

ITY

OF

DE

ND

RIT

ICC

EL

LS

TO

PR

OC

ES

SA

ND

PR

ES

EN

TA

NT

IGE

NS

FR

OM

VA

CU

OL

AR

PA

TH

OG

EN

S

macrophages, which contain the restrictive Lgn1 allele, found that there

appears to be multifactorial resistance to L. pneumophila. This resistance is

mediated in part through retrafficking of the L. pneumophila phagosome to

the lysosome and the induction of apoptosis in infected cells (Derre and

Isberg, 2004).

7.4 INNATE IMMUNE RESPONSES TO LEGIONELLABY TOLL-LIKE RECEPTORS

Work examining the ability of L. pneumophila lipopolysaccharide (LPS) to

activate TLR signaling in vitro indicates that TLR2 rather than TLR4 is

primarily responsible for initiating cellular responses to L. pneumophila LPS

(Girard et al., 2003). Highly purified LPS is able to activate TLR2 signaling

in macrophages, while TLR4-deficient macrophages show defects in their

ability to respond to L. pneumophila LPS (Girard et al., 2003). This result

is supported by data showing that intracellular growth of L. pneumophila

is enhanced within TLR2�/� macrophages compared to WT and TLR4�/�

macrophages (Akamine et al., 2005). This increase in intracellular growth

is accompanied by a significant decrease in the production of IL-12,

which is essential for clearing L. pneumophila infection (Akamine et al.,

2005). These studies indicate that TLR2, rather than TLR4, plays a principal

role in initiating immune responses to L. pneumophila. However, the

importance of TLR2 in controlling Legionella replication in vivo is not yet

established.

TLR5 has also been implicated in the innate immune response

to L. pneumophila. TLR5 is responsible for sensing bacterial flagellin

(Smith et al., 2003). L. pneumophila flagellin activates TLR5 signaling

pathways, resulting in the induction of IL-8 production by lung epithelial

cell lines (Hawn et al., 2003). Additionally, epidemiological studies provide

a link between a common human genetic polymorphism, which produces

a truncated TLR5 protein that cannot respond to flagellin, and susceptibility

to L. pneumophila-induced pneumonia in humans (Hawn et al., 2003).

Whether TLR5 is required for the initiation of the immune response

to L. pneumophila is unclear. It would be expected that other TLRs, such

as TLR9, which recognizes bacterial CpG DNA, would also be involved.

Although the role of the TLRs in initiating the immune response to

L. pneumophila infection has not yet been addressed, it is expected

that some of the TLRs would be involved in the initiation of cytokine

production.

�

144

S.

SH

IN,

C.

NO

GU

EIR

AA

ND

C.R

.R

OY

7.5 INDUCTION OF CYTOKINES FOLLOWING

LEGIONELLA INFECTION

Humans infected with L. pneumophila develop a predominantly T1 cytokine

profile, which mimics the Th1 cytokine response (Tateda et al., 1998).

The T1/T2 cytokine balance in infected hosts appears to be critical

for the outcome of L. pneumophila infection. In mice, neutrophils have

been shown to play a prominent role in determining subsequent T1/T2

host responses to L. pneumophila (Tateda et al., 2001). Depletion of

neutrophils results in a hundred-fold increase in susceptibility of mice

to lethal pneumonia. This effect was not due to direct neutrophil killing

of L. pneumophila. Instead, neutrophils appeared to modulate the

cytokine profile of infected mice. These mice had a significant decrease

in IFN-g and IL-12 production and an increase in the T2 cytokines IL-4

and IL-10 (Tateda et al., 2001). Additionally, IL-10 treatment can enhance

the growth of L. pneumophila in human monocytes and reverse the

protective effect of IFN-g (Park and Skerrett, 1996). This indicates that

development of a T1 response is required for clearance of L. pneumophila

infection.

IFN-g is a primary T1 cytokine and is critical in the activation of

macrophages. It is required for the resolution of infections caused by

a variety of bacteria, such as Salmonella typhimurium and Mycobacterium

tuberculosis. IFN-g is also critical for the clearance of L. pneumophila

infection in mice. Mice depleted of IFN-g are unable to clear L. pneumophila

infection (Brieland et al., 1994). This is supported by experiments demon-

strating that mice genetically deficient in IFN-g are also unable to clear

infection (Heath et al., 1996; Shinozawa et al., 2002). Instead, they develop

persistent and replicative L. pneumophila infections, which disseminate

to the spleen (Heath et al., 1996). In vivo, natural killer cells appear to

be the primary source of IFN-g production during L. pneumophila infection

(Blanchard et al., 1988; Deng et al., 2001). IFN-g is also induced in non-

permissive macrophages following in vitro L. pneumophila infection (Salins

et al., 2001).

7.6 ADAPTIVE IMMUNE RESPONSES TO LEGIONELLA INFECTION

Studies examining the immune response to L. pneumophila have

revealed that adaptive immunity develops during the course of infection.

Humans infected with L. pneumophila often produce a specific humoral

response. Infected mice also develop a humoral response to L. pneumophila.

�145

PE

CU

LIA

RA

BIL

ITY

OF

DE

ND

RIT

ICC

EL

LS

TO

PR

OC

ES

SA

ND

PR

ES

EN

TA

NT

IGE

NS

FR

OM

VA

CU

OL

AR

PA

TH

OG

EN

S

Infection of A/J mice results in the recruitment of B lymphocytes into

the lung and the production of anti-L. pneumophila antibodies (Brieland

et al., 1996). Additionally, guinea pigs sublethally infected with

L. pneumophila also develop a robust humoral response (Breiman and

Horwitz, 1987). However, the precise role played by the antibody response

in clearing infection or in conferring a protective immune response is

currently unclear. In fact, it has been reported that antibodies may actually

promote L. pneumophila infection by increasing uptake of L. pneumophila

in macrophages through opsonization (Horwitz and Silverstein, 1981).

Several studies indicate that L. pneumophila-infected animals develop

an acquired Th1 immune response, which appears to result in the clear-

ance of infection and in the development of a memory T cell response.

Splenic lymphocytes from L. pneumophila-infected guinea pigs proliferate

robustly in response to L. pneumophila antigens in vitro (Breiman and

Horwitz, 1987). Guinea pigs also develop delayed-type hypersensitivity

(DTH), a classic sign of Th1 immunity, which results in an inflammatory

reaction at the site of intercutaneous infection of L. pneumophila (Weeratna

et al., 1994). The role of T cells during a primary L. pneumophila infection

has been examined by depletion of either CD4, CD8 or both T cell subsets

(Susa et al., 1998). Mice depleted of CD4 or CD8 T cells show a slight

delay in clearance of L. pneumophila, whereas mice depleted of both CD4

and CD8 T cells are significantly impaired in their ability to control

L. pneumophila infection, resulting in increased lethality (Susa et al., 1998).

This indicates a critical role for both CD4 and CD8 T cells in control of

L. pneumophila infection. However, it is unclear whether this primary

T cell response is directed mainly against L. pneumophila antigens or

whether it is due to an intrinsic requirement for T cell cytokine production.

7.7 RESTRICTION OF LEGIONELLA GROWTH IN ACTIVATED

MACROPHAGES

It has long been known that treating macrophages with IFN-g enhances

their ability to inhibit the growth of many intracellular bacterial pathogens.

Similarly, IFN-g also restricts L. pneumophila growth in macrophages

(Bhardwaj et al., 1986; Nash et al., 1988). In the case of L. pneumophila, it is

thought that intracellular growth restriction is mediated in part by restricting

the availability of intracellular iron (Byrd and Horwitz, 1989). L. pneumophila

growth within macrophages normally requires the presence of iron, which is

transported by the transferrin receptor (Byrd and Horwitz, 1989).

Macrophages activated by IFN-g downregulate expression of transferrin

�

146

S.

SH

IN,

C.

NO

GU

EIR

AA

ND

C.R

.R

OY

receptor, which limits the amount of iron and thus inhibits L. pneumophila

growth (Byrd and Horwitz, 1989). Addition of iron to IFN-g treated

macrophages can reverse the restriction of L. pneumophila replication

(Byrd and Horwitz, 1991).

Macrophages activated with IFN-g are also able to override the ability

of pathogens such as Mycobacterium avium to alter trafficking of their

phagosomes, resulting in phagolysosome fusion (Schaible et al., 1998).

This appears to be the case for L. pneumophila as well. In IFN-g activated

macrophages, the L. pneumophila phagosome appears to mature into

a phagolysosome, as evidenced by the acquisition of the lysosomal

markers LAMP-2 and cathepsin D (Santic et al., 2005). The mechanism

of how IFN-g is able to initiate the retrafficking of the L. pneumophila

phagosome is unknown.

In addition, the ability of IFN-g-treated macrophages to inhibit

L. pneumophila growth may also be mediated by the production of reactive

nitric oxide or reactive oxygen intermediates, both of which are essential

anti-microbial effectors of macrophages. However, the importance of nitric

oxide and oxygen intermediates in controlling L. pneumophila infection

is unclear. Treatment of activated macrophages with superoxide dismutase

and catalase does not affect the inhibition of L. pneumophila growth,

suggesting that reactive oxygen intermediates are dispensable for control-

ling Legionella replication (Klein et al., 1991). Treatment of A/J mice

with the NO synthetase inhibitor N-monomethyl L-arginine (L-MMA)

inhibits their ability to clear L. pneumophila infection (Brieland et al.,

1995). However, treatment of BALB/c macrophages or mice with L-MMA

does not significantly inhibit clearance of L. pneumophila infection (Heath

et al., 1996; Yamamoto et al., 1996). Using C57BL/6 mice genetically

deficient for NADPH oxidase, it was found that NADPH oxidase appears

to be important in early control of L. pneumophila infection, but is

dispensable at later time points (Saito et al., 2001). Collectively, these

data indicate that the role of nitric oxide intermediates in controlling

L. pneumophila growth may differ between susceptible and resistant

macrophages. These results also indicate that mechanisms other than

the nitric oxide burst are also responsible for controlling L. pneumophila

replication. It is possible that reactive oxygen and nitric oxide inter-

mediates may have redundant roles in inhibiting L. pneumophila growth.

This possibility has not yet been formally tested.

In addition to IFN-g, type I IFN (IFN-ab) treatment of permissive

macrophages results in an inhibition of intracellular L. pneumophila repli-

cation (Schiavoni et al., 2004). Depletion of IFN-ab from non-permissive

�147

PE

CU

LIA

RA

BIL

ITY

OF

DE

ND

RIT

ICC

EL

LS

TO

PR

OC

ES

SA

ND

PR

ES

EN

TA

NT

IGE

NS

FR

OM

VA

CU

OL

AR

PA

TH

OG

EN

S

macrophage cultures enhances their susceptibility to L. pneumophila

infection, indicating a role for endogenous IFN-ab in restricting intra-

cellular L. pneumophila growth (Schiavoni et al., 2004). IFN-ab appears

to act independently of IFN-g (Schiavoni et al., 2004). The molecular

mechanisms underlying how IFN-ab controls intracellular L. pneumophila

replication and whether it is through a mechanism similar to that used

by IFN-g is unknown.

7.8 LEGIONELLA GROWTH RESTRICTION BY MURINE

DENDRITIC CELLS

L. pneumophila has been used as a model organism to dissect the

mechanisms by which immune responses are generated against vacuolar

pathogens. To understand how immunity against vacuolar pathogens

is generated, it is important to determine how T cell responses are primed

and the mechanisms by which bacterial antigens become available for

presentation on MHC class II. Although the interactions between

L. pneumophila and macrophages have been extensively studied over

the past years, the mechanisms underlying the development of adaptive

immunity to this organism remain elusive (Kikuchi et al., 2004). Recent

studies have revealed that DCs are also likely to play an important role in

the initiation of the adaptive immune response to L. pneumophila (Neild

and Roy, 2003).

Using DC derived from the permissive A/J mouse strain, it was

determined that the intracellular behavior of L. pneumophila inside

dendritic cells (DCs) differs from that in macrophages. Specifically, in

contrast to macrophages, DCs inhibit replication of Legionella. Twelve

hours after infecting DCs with L. pneumophila, the number of vacuoles

harboring replicating L. pneumophila, as determined by the vacuole having

more than two bacterial cells, is extremely low. By contrast, when the

same experiment is performed in macrophages, roughly 75 per cent of

all vacuoles containing L. pneumophila harbor more than four bacteria due

to robust intracellular growth (Neild and Roy, 2004). No significant host

cell killing was observed after infection of DCs by wild type L. pneumophila,

indicating that the DCs restrict the growth of L. pneumophila by a

mechanism independent of cell death (Neild and Roy, 2004).

To better understand how DCs restrict the growth of L. pneumophila,

morphological studies were conducted to determine whether vacuoles

containing bacteria traffic differently in DCs compared to macrophages.

These studies showed that vacuoles containing L. pneumophila traffic to

�

148

S.

SH

IN,

C.

NO

GU

EIR

AA

ND

C.R

.R

OY

the ER in both DCs and macrophages, forming similar ER-derived vacuoles.

ER vesicles, ribosomes and mitochondria are associated with the mem-

brane of the vacuole established by L. pneumophila, in contrast to the

naked membrane vacuole containing dotA strains which fuses with the

lysosome. Inside DCs, L. pneumophila cannot replicate but it remains

metabolically active (Neild and Roy, 2004). Thus, the mechanism by

which DCs restrict growth of L. pneumophila occurs after establishment

of the ER vacuole and after L. pneumophila begins to respond to this

new environment.

7.9 PRESENTATION OF LEGIONELLA ANTIGENS BY

INFECTED DENDRITIC CELLS

Although L. pneumophila reside within an ER-derived compartment in DCs,

bacterial antigens are still presented on MHC class II molecules. DCs

infected with L. pneumophila stimulate immune CD4þ T cells, indicating

that L. pneumophila antigens are being processed and presented on MHC

class II (Neild and Roy, 2003). Interestingly, in vivo studies show that

after immunization of mice with wild type or dotA mutant L. pneumophila,

two sets of T cell responses are generated: those that can respond to

common determinants presented by both wild-type and dotA L. pneumophila

and another subset of T cells that seem specific for bacterial determinants

synthesized and presented after wild-type L. pneumophila establish an

ER-derived vacuole (Neild and Roy, 2004). These data reveal that DCs

have the ability to restrict the intracellular growth of L. pneumophila

and present bacteria antigens on MHC II, which would be expected to be

an important property for priming effective T-cell mediated responses to

vacuolar pathogens.

In a second study, it was shown that DCs pulsed with dead

L. pneumophila, but not with live bacteria, undergo maturation, resulting

in the upregulation of MHC II, costimulatory, adhesion and signaling

molecules such as CD40, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2)

(Kikuchi et al., 2004). Apart from IL-1b, the same event is also true

for proinflammatory cytokine expression, including IL-12, TNF-a and

IL-6. Moreover, when adoptively transferred, dead L. pneumophila-

pulsed DCs induce L. pneumophila-specific protective immunity in a

manner dependent on MHC II Ag presentation to CD4þ T cells (Kikuchi

et al., 2004).

These findings provide a better understanding of the role of DCs

and the critical link between the innate and adaptive immune responses

�149

PE

CU

LIA

RA

BIL

ITY

OF

DE

ND

RIT

ICC

EL

LS

TO

PR

OC

ES

SA

ND

PR

ES

EN

TA

NT

IGE

NS

FR

OM

VA

CU

OL

AR

PA

TH

OG

EN

S

to vacuolar pathogens. The ability of phagocytes to restrict L. pneumophila

growth appears to be the key to host immunity (Figure 7.1). However,

the mechanisms by which DCs restrict L. pneumophila intracellular

growth are unknown. A number of studies show that control of iron

availability is required for restricting L. pneumophila growth in activated

macrophages but it still remains unclear which host gene products are

involved in restriction of intracellular L. pneumophila growth in both

macrophages and DCs (Byrd and Horwitz, 1989; Neild and Roy, 2004). It is

also unknown whether the ability of DCs to restrict bacterial repli-

cation depends upon the upregulation of DC effector functions, or

whether the restriction of L. pneumophila growth by DCs occurs indepen-

dently of DC maturation. Thus, it is currently of great interest to identify

the potential genes involved in restricting L. pneumophila replication

within DCs.

7.10 FUTURE QUESTIONS IN LEGIONELLA IMMUNITY

It is becoming increasingly clear that DCs and macrophages have

mechanisms for sensing L. pneumophila products in the cytosol and that

detection of these products can limit growth of the bacterium. During the

establishment of the L. pneumophila vacuole, the L. pneumophila Dot/Icm

type IV secretion system actively transports bacterial products into the

host cytosol (Nagai et al., 2002; Chen et al., 2004; Luo and Isberg, 2004). Some

of these bacterial components are most likely sensed by members of the

Nod-LRR protein family. In particular, the Nod-LRR family member Birc1e,

which has been mapped as a genetic susceptibility locus for L. pneumophila

infection, is thought to be involved in the detection of L. pneumophila

infection (Diez et al., 2003; Wright et al., 2003). Birc1e has been shown to

be involved in restricting the growth of L. pneumophila inside macrophages

(Diez et al., 2003; Wright et al., 2003; Derre and Isberg, 2004). How Birc1e

accomplishes this is unclear, but it is expected that Birc1e regulates an

intracellular effector pathway which controls L. pneumophila replication

and trafficking. The role of other Nod-LRR proteins in the immune response

to L. pneumophila has yet to be determined. It is possible that Birc1e or

other Nod-LRR proteins are involved in the restriction of L. pneumophila

growth in DCs as well.

In addition to Birc1e, other host factors such as IFN-g activation, iron

sequestration and reactive oxygen and nitric oxide intermediates have

been proposed to play a role in limiting L. pneumophila replication in

macrophages. Further work needs to be done on whether these factors

�

150

S.

SH

IN,

C.

NO

GU

EIR

AA

ND

C.R

.R

OY

�

Fig

ure

7.1

.M

ech

anis

ms

of

def

ence

init

iate

daf

terLegionella

infe

ctio

n.

Fo

llo

win

gLegionella

infe

ctio

nth

etw

oar

ms

of

the

inn

ate

and

adap

tive

imm

un

e

resp

on

sear

ein

itia

ted

.a.C

lear

ance

by

the

Inn

ate

Imm

un

eS

yste

m(u

pp

erp

anel

).Legionella

infe

ctio

nst

imu

late

sm

acro

ph

age

TL

Rs

and

NO

D-L

RR

s.T

his

lead

s

toth

ep

rod

uct

ion

of

pro

infl

amm

ato

rycy

tok

ines

(IL

-12

and

IL-18)

by

mac

rop

hag

es.N

Kce

lls

are

recr

uit

edto

the

site

ofin

fect

ion

and

pro

du

ceIF

N-g

.Res

tric

tio

n

of

intr

acel

lula

rre

pli

cati

onLegionella

by

IFN

-gac

tiva

ted

mac

rop

hag

es.

b.

Ind

uct

ion

of

Ad

apti

veIm

mu

nit

y(l

ow

erp

anel

).Im

mat

ure

DC

sre

stri

ctLegionella

rep

lica

tio

nan

dar

ein

du

ced

tom

atu

rati

on

.M

atu

reD

Cs

stim

ula

ten

aive

Tce

lls

wit

hre

cep

tors

spec

ific

forLegionella

anti

gen

s.N

aive

Tce

lls

bec

om

eT

h-1

typ

e

effe

cto

rsth

atca

np

rod

uce

IFN

-gin

resp

on

seto

Legionella

anti

gen

sd

isp

laye

do

nth

esu

rfac

eo

fin

fect

edm

acro

ph

ages

.

are also utilized by DCs to control L. pneumophila growth. Most likely,

similar mechanisms are utilized by both DCs and activated macrophages

to control L. pneumophila growth. A comparative proteomic analysis of

the L. pneumophila phagosome in DCs, IFN-g-activated macrophages

and non-activated macrophages may identify novel host factors involved

in the restriction of L. pneumophila growth. Understanding how murine

macrophages and dendritic cells are able to restrict the intracellular growth

of L. pneumophila will also provide insight into how the immune

response is able to control other intracellular pathogens such as Chlamydia

trachomatis and Mycobacterium tuberculosis.

Animals infected with L. pneumophila can develop a robust and pro-

tective T cell response. Development of a T cell response would require

the presentation of L. pneumophila antigens on MHC class I and II

molecules. How processing and presentation of L. pneumophila antigens on

MHC molecules occurs in light of the observation that the L. pneumophila

vacuole is distinct from the endocytic pathway is unknown. We know

that DCs are able to inhibit the intracellular replication of L. pneumophila,

yet L. pneumophila is not destroyed via lysosomal fusion (Neild and Roy,

2003). Although L. pneumophila is able to reside in a phagosome that

inhibits fusion with lysosomes, DCs infected with L. pneumophila are

capable of eliciting a L. pneumophila-specific CD4 T cell response (Neild

and Roy, 2003). This raises the question of how L. pneumophila antigens

are processed and presented on MHC class II molecules. Perhaps MHC

class II processing and presentation stems from the L. pneumophila

phagosome. Alternatively, L. pneumophila antigens may be able to leave

the phagosome and enter the MHC class II presentation pathway

elsewhere in the cell.

As for MHC class I presentation, it would be expected that

L. pneumophila antigens can become accessible to the MHC class I

presentation pathway via the transport of L. pneumophila proteins through

the Dot/Icm type IV secretory apparatus into the cytosol, where they are

then processed by the proteasome and transported via TAP into the ER

for loading onto MHC class I molecules. Alternatively, L. pneumophila

antigens might be processed by alternative pathways, such as the cross-

presentation pathway, which would require the host cell-mediated trans-

port of the lumenal contents of the phagosome and their processing and

loading onto MHC class I. Answering these questions will most likely

require the identification of the L. pneumophila antigens that are being

presented. Studying how L. pneumophila antigens become accessible to the

MHC class I and II pathways most likely will reveal novel antigen processing

�

152

S.

SH

IN,

C.

NO

GU

EIR

AA

ND

C.R

.R

OY

and presentation pathways. Additionally, it may provide clues into how

the antigens of other vacuolar pathogens are processed as well.

The ease of genetic manipulation of Legionella along with the existence

of known mutants with defects in their ability to modify phagosomal

trafficking provides an excellent system to examine how immune

responses are generated against bacteria in distinct subcellular compart-

ments. Furthermore, the recent observations on the differential ability

of macrophages and DCs to control Legionella growth provides investi-

gators with the ability to dissect both the host and bacterial sides of

the Legionella�phagocyte interaction. These studies will doubtless lead

to new insights into the mechanisms of how phagocytes control replication

of bacterial pathogens.

REFERENCES

Akamine, M., F. Higa et al. (2005). Differential roles of Toll-like receptors 2

and 4 in in vitro responses of macrophages to Legionella pneumophila.

Infect. Immun. 73(1), 352�61.

Bhardwaj, N., T. W. Nash et al. (1986). Interferon-gamma-activated human

monocytes inhibit the intracellular multiplication of Legionella pneumophila.

J. Immunol. 137(8), 2662�9.

Blanchard, D. K., H. Friedman et al. (1988). Role of gamma interferon

in induction of natural killer activity by Legionella pneumophila in vitro

and in an experimental murine infection model. Infect. Immun.

56(5), 1187�93.

Breiman, R. F. and M. A. Horwitz (1987). Guinea pigs sublethally infected

with aerosolized Legionella pneumophila develop humoral and cell-mediated

immune responses and are protected against lethal aerosol challenge.

A model for studying host defense against lung infections caused by

intracellular pathogens. J. Exp. Med. 165(3), 799�811.

Brenner, D. J., A. G. Steigerwalt et al. (1979). Classification of the Legionnaires’

disease bacterium: Legionella pneumophila, genus novum, species nova,

of the family Legionellaceae, familia nova. Ann. Intern. Med. 90(4), 656�8.

Brieland, J., P. Freeman et al. (1994). Replicative Legionella pneumophila

lung infection in intratracheally inoculated A/J mice. A murine model of

human Legionnaires’ disease. Am. J. Pathol. 145(6), 1537�46.

Brieland, J. K., L. A. Heath et al. (1996). Humoral immunity and

regulation of intrapulmonary growth of Legionella pneumophila in

the immunocompetent host. J. Immunol. 157(11), 5002�8.

�153

PE

CU

LIA

RA

BIL

ITY

OF

DE

ND

RIT

ICC

EL

LS

TO

PR

OC

ES

SA

ND

PR

ES

EN

TA

NT

IGE

NS

FR

OM

VA

CU

OL

AR

PA

TH

OG

EN

S

Brieland, J. K., D. G. Remick et al. (1995). In vivo regulation of replicative

Legionella pneumophila lung infection by endogenous tumor necrosis factor

alpha and nitric oxide. Infect. Immun. 63(9), 3253�8.

Byrd, T. F. and M. A. Horwitz (1989). Interferon gamma-activated human

monocytes downregulate transferrin receptors and inhibit the intracellular

multiplication of Legionella pneumophila by limiting the availability of iron.

J. Clin. Invest. 83(5), 1457�65.

Byrd, T. F. and M. A. Horwitz (1991). Lactoferrin inhibits or promotes

Legionella pneumophila intracellular multiplication in nonactivated and

interferon gamma-activated human monocytes depending upon its degree

of iron saturation. Iron-lactoferrin and nonphysiologic iron chelates

reverse monocyte activation against Legionella pneumophila. J. Clin. Invest.

88(4), 1103�12.

Chen, J., K. S. de Felipe et al. (2004). Legionella effectors that promote nonlytic

release from protozoa. Science 303(5662), 1358�61.

Deng, J. C., K. Tateda et al. (2001). Transient transgenic expression of gamma

interferon promotes Legionella pneumophila clearance in immunocompetent

hosts. Infect. Immun. 69(10), 6382�90.

Derre, I. and R. R. Isberg (2004). Macrophages from mice with the restrictive

Lgn1 allele exhibit multifactorial resistance to Legionella pneumophila.

Infect. Immun. 72(11), 6221�9.

Diez, E., S. H. Lee et al. (2003). Birc1e is the gene within the Lgn1 locus associated

with resistance to Legionella pneumophila. Nat. Genet. 33(1), 55�60.

Fields, B. S. (1996). The molecular ecology of Legionellae. Trends Microbiol. 4(7),

286�90.

Fliermans, C. B., W. B. Cherry et al. (1981). Ecological distribution of Legionella

pneumophila. Appl. Environ. Microbiol. 41(1), 9�16.

Fraser, D. W., T. R. Tsai et al. (1977). Legionnaires’ disease: description of

an epidemic of pneumonia. N. Engl. J. Med. 297(22), 1189�97.

Girard, R., T. Pedron et al. (2003). Lipopolysaccharides from Legionella and

Rhizobium stimulate mouse bone marrow granulocytes via Toll-like

receptor 2. J. Cell Sci. 116(Pt 2), 293�302.

Hawn, T. R., A. Verbon et al. (2003). A common dominant TLR5 stop

codon polymorphism abolishes flagellin signaling and is associated

with susceptibility to legionnaires’ disease. J. Exp. Med. 198(10),

1563�72.

Heath, L., C. Chrisp et al. (1996). Effector mechanisms responsible for

gamma interferon-mediated host resistance to Legionella pneumophila

lung infection: the role of endogenous nitric oxide differs in susceptible

and resistant murine hosts. Infect. Immun. 64(12), 5151�60.

�

154

S.

SH

IN,

C.

NO

GU

EIR

AA

ND

C.R

.R

OY

Horwitz, M. A. (1983). Formation of a novel phagosome by the Legionnaires’

disease bacterium (Legionella pneumophila) in human monocytes. J. Exp.

Med. 158(4), 1319�31.

Horwitz, M. A. (1987). Characterization of avirulent mutant Legionella

pneumophila that survive but do not multiply within human monocytes.

J. Exp. Med. 166(5), 1310�28.

Horwitz, M. A. and S. C. Silverstein (1981). Interaction of the legionnaires’

disease bacterium (Legionella pneumophila) with human phagocytes.

II. Antibody promotes binding of L. pneumophila to monocytes but does

not inhibit intracellular multiplication. J. Exp. Med. 153(2), 398�406.

Inohara, N., M. Chamaillard et al. (2005). NOD-LRR proteins: role in

host�microbial interactions and inflammatory disease. Annu. Rev.

Biochem. 74: 355�83.

Kagan, J. C. and C. R. Roy (2002). Legionella phagosomes intercept

vesicular traffic from endoplasmic reticulum exit sites. Nat. Cell Biol.

4(12), 945�54.

Kikuchi, T., T. Kobayashi et al. (2004). Dendritic cells pulsed with live and

dead Legionella pneumophila elicit distinct immune responses. J. Immunol.

172(3), 1727�34.

Klein, T. W., Y. Yamamoto et al. (1991). Interferon-gamma induced resistance

to Legionella pneumophila in susceptible A/J mouse macrophages. J. Leukoc.

Biol. 49(1), 98�103.

Langmark, J., M. V. Storey et al. (2005). Accumulation and fate of

microorganisms and microspheres in biofilms formed in a pilot-scale

water distribution system. Appl. Environ. Microbiol. 71(2), 706�12.

Luo, Z. Q. and R. R. Isberg (2004). Multiple substrates of the

Legionella pneumophila Dot/Icm system identified by interbacterial

protein transfer. Proc. Natl Acad. Sci. U S A 101(3), 841�6.

Marra, A., S. J. Blander et al. (1992). Identification of a Legionella pneumophila

locus required for intracellular multiplication in human macrophages.

Proc. Natl Acad. Sci. U S A 89(20), 9607�11.

McDade, J. E., C. C. Shepard et al. (1977). Legionnaires’ disease: isolation

of a bacterium and demonstration of its role in other respiratory disease.

N. Engl. J. Med. 297(22), 1197�203.

Nagai, H., J. C. Kagan et al. (2002). A bacterial guanine nucleotide

exchange factor activates ARF on Legionella phagosomes. Science

295(5555), 679�82.

Nash, T. W., D. M. Libby et al. (1988). IFN-gamma-activated human

alveolar macrophages inhibit the intracellular multiplication

of Legionella pneumophila. J. Immunol. 140(11), 3978�81.

�155

PE

CU

LIA

RA

BIL

ITY

OF

DE

ND

RIT

ICC

EL

LS

TO

PR

OC

ES

SA

ND

PR

ES

EN

TA

NT

IGE

NS

FR

OM

VA

CU

OL

AR

PA

TH

OG

EN

S

Neild, A. L. and C. R. Roy (2003). Legionella reveal dendritic cell functions

that facilitate selection of antigens for MHC class II presentation.

Immunity 18(6), 813�23.

Neild, A. L. and C. R. Roy (2004). Immunity to vacuolar pathogens: what

can we learn from Legionella? Cell Microbiol. 6(11), 1011�18.

Park, D. R. and S. J. Skerrett (1996). IL-10 enhances the growth of

Legionella pneumophila in human mononuclear phagocytes and reverses

the protective effect of IFN-gamma: differential responses of blood

monocytes and alveolar macrophages. J. Immunol. 157(6), 2528�38.

Roy, C. R. and L. G. Tilney (2002). The road less traveled: transport of Legionella to

the endoplasmic reticulum. J. Cell Biol. 158(3), 415�19.

Saito, M., H. Kajiwara et al. (2001). Fate of Legionella pneumophila in

macrophages of C57BL/6 chronic granulomatous disease mice.

Microbiol. Immunol. 45(7), 539�41.

Salins, S., C. Newton et al. (2001). Differential induction of gamma

interferon in Legionella pneumophila-infected macrophages from BALB/c

and A/J mice. Infect. Immun. 69(6), 3605�10.

Santic, M., M. Molmeret et al. (2005). Maturation of the Legionella

pneumophila-containing phagosome into a phagolysosome within

gamma interferon-activated macrophages. Infect. Immun. 73(5), 3166�71.

Schaible, U. E., S. Sturgill-Koszycki et al. (1998). Cytokine activation leads

to acidification and increases maturation of Mycobacterium

avium-containing phagosomes in murine macrophages. J. Immunol.

160(3), 1290�6.

Schiavoni, G., C. Mauri et al. (2004). Type I IFN protects permissive macrophages

from Legionella pneumophila infection through an IFN-gamma-independent

pathway. J. Immunol. 173(2), 1266�75.

Segal, G., M. Purcell et al. (1998). Host cell killing and bacterial conjugation

require overlapping sets of genes within a 22-kb region of the

Legionella pneumophila genome. Proc. Natl Acad. Sci. U S A 95(4),

1669�74.

Shinozawa, Y., T. Matsumoto et al. (2002). Role of interferon-gamma in

inflammatory responses in murine respiratory infection with Legionella

pneumophila. J. Med. Microbiol. 51(3), 225�30.

Smith, K. D., E. Andersen-Nissen et al. (2003). Toll-like receptor 5 recognizes

a conserved site on flagellin required for protofilament formation and

bacterial motility. Nat. Immunol. 4(12), 1247�53.

Susa, M., B. Ticac et al. (1998). Legionella pneumophila infection in

intratracheally inoculated T cell-depleted or -nondepleted A/J mice.

J. Immunol. 160(1), 316�21.

�

156

S.

SH

IN,

C.

NO

GU

EIR

AA

ND

C.R

.R

OY

Szymanska, J., L. Wdowiak et al. (2004). Microbial quality of water in dental

unit reservoirs. Ann. Agric. Environ. Med. 11(2), 355�8.

Tateda, K., T. Matsumoto et al. (1998). Serum cytokines in patients with

Legionella pneumonia: relative predominance of Th1-type cytokines.

Clin. Diagn. Lab. Immunol. 5(3), 401�3.

Tateda, K., T. A. Moore et al. (2001). Early recruitment of neutrophils

determines subsequent T1/T2 host responses in a murine model of

Legionella pneumophila pneumonia. J. Immunol. 166(5), 3355�61.

Tilney, L. G., O. S. Harb et al. (2001). How the parasitic bacterium Legionella

pneumophila modifies its phagosome and transforms it into rough ER:

implications for conversion of plasma membrane to the ER membrane.

J. Cell Sci. 114(Pt 24), 4637�50.

Ting, J. P. and K. L. Williams (2005). The CATERPILLER family: An ancient

family of immune/apoptotic proteins. Clin. Immunol. 115(1), 33�7.

Vogel, J. P., H. L. Andrews et al. (1998). Conjugative transfer by the

virulence system of Legionella pneumophila. Science 279(5352), 873�6.

Weeratna, R., D. A. Stamler et al. (1994). Human and guinea pig immune

responses to Legionella pneumophila protein antigens OmpS and Hsp60.

Infect. Immun. 62(8), 3454�62.

Wright, E. K., S. A. Goodart et al. (2003). Naip5 affects host susceptibility to

the intracellular pathogen Legionella pneumophila. Curr. Biol. 13(1), 27�36.

Yamamoto, Y., T. W. Klein et al. (1996). Immunoregulatory role of nitric oxide

in Legionella pneumophila-infected macrophages. Cell. Immunol. 171(2),

231�9.

Yamamoto, Y., T. W. Klein et al. (1988). Growth of Legionella pneumophila

in thioglycolate-elicited peritoneal macrophages from A/J mice.

Infect. Immun. 56(2), 370�5.

Yee, R. B. and R. M. Wadowsky (1982). Multiplication of Legionella pneumophila in

unsterilized tap water. Appl. Environ. Microbiol. 43(6), 1330�4.

�157

PE

CU

LIA

RA

BIL

ITY

OF

DE

ND

RIT

ICC

EL

LS

TO

PR

OC

ES

SA

ND

PR

ES

EN

TA

NT

IGE

NS

FR

OM

VA

CU

OL

AR

PA

TH

OG

EN

S

CHAPTER 8

Dendritic cells, macrophages andcross-presentation of bacterial antigens:a lesson from Salmonella

Mary Jo WickGoteborg University

8.1 INTRODUCTION

Immunity to a bacterial pathogen requires the generation of bacteria-

specific T cells with appropriate effector function. Eliciting T cells during

infection requires internalization of the bacteria and processing of

bacterial proteins to generate peptides for presentation on major histocom-

patibility complex (MHC) class I (MHC-I) and/or MHC class II (MHC-II)

molecules, depending on the pathogen. As not all host cells have the

capacity to phagocytose bacteria, and not all bacterial pathogens have the

capacity to actively invade non-phagocytic cells, phagocytic antigen present-

ing cells, macrophages and immature dendritic cells (DCs), are the key

players in generating adaptive immunity to bacteria.

Both macrophages and immature dendritic cells can present antigens

from the bacteria they internalize on their own MHC-I and MHC-II

molecules and thus carry out so-called direct presentation of bacterial

antigens (Sundquist et al., 2004; Harding et al., 2003). Direct presentation

of bacterial antigens on MHC-II is the expected outcome following

phagocytosis of bacteria and is the event necessary to elicit CD4þ T cells.

However, both macrophages and DCs can also present antigens from

internalized bacteria on MHC-I, molecules most renowned for their

presentation of peptides derived from endogenously synthesized proteins

(Rock and Goldberg, 1999), and generate CD8þ Tcells (Sundquist et al., 2004;

Harding et al., 2003).

Given the capacity of both macrophages and DCs to directly present

bacterial antigens on MHC-I and MHC-II, these cells in principle could

initiate adaptive immunity during primary infection. However, it is only

DCs that have this ability (Banchereau and Steinman, 1998). This is due

to their ability to migrate to secondary lymphoid organs and interact

with naive T cells in addition to their antigen presentation capacity.

Dendritic cells also have sufficient expression of MHC and costimulatory

molecules to activate naive T cells. Thus, DCs fulfill a unique niche in anti-

bacterial immunity as the key phagocytic cells that start an adaptive

immune response during infection.

This chapter summarizes data relating to presentation of antigens

derived from the facultative intracellular bacteria Salmonella enterica

serovar typhimurium (S. typhimurium) by DCs. Particular emphasis will

be on antigen presentation on MHC-I. In addition to discussing direct

presentation of antigens from Salmonella-infected cells, presentation of

bacterial antigens by cells that are not themselves infected will also be

discussed. That is, a non-infected DC can acquire antigenic material from

other infected cells that die as a consequence of Salmonella in a process

called indirect or cross-presentation (see also Chapter 3). Cross-presentation

of bacterial antigens by bystander DCs will also be summarized. Before

discussing antigen presentation, a brief overview of Salmonella infection

will be given.

8.2 SALMONELLA INFECTION AND SURVIVAL IN HOST CELLS

S. typhimurium is an enteric pathogen naturally acquired orally in

contaminated food or water. This Gram-negative facultative intracellular

bacterium penetrates the intestinal epithelium, initially seeding Peyer’s

patches, and being detected thereafter in deeper tissues including the

mesenteric lymph nodes, spleen and liver (Sundquist and Wick, 2005;

McSorley et al., 2002). In addition to M cell-mediated traversal of bacteria

across the intestinal epithelium, which appears to be the predominant

pathway used by invasive Salmonella to cross the gut barrier (Niess

et al., 2005; Jones et al., 1994), intestinal DCs seem to also be involved in

the initial traversal of Salmonella across the intestine (Niess et al., 2005;

Macpherson and Uhr, 2004; Rescigno et al., 2001). This is due to their ability

to sample luminal Salmonella by extending their dendrites between epithelial

cells and transport the bacteria across the epithelial layer (Niess et al., 2005;

Macpherson and Uhr, 2004; Rescigno et al., 2001).

Once Salmonella penetrate the intestinal barrier, cells of the innate

immune system initiate an inflammatory response and direct the adap-

tive immune response (Mastroeni, 2002). Salmonella invasion results in

infiltration of neutrophils and monocytes/macrophages, as well as DCs,

to infected organs (Sundquist and Wick, 2005; Kirby et al., 2001, 2002;

�

160

M.

J.W

ICK

Mastroeni, 2002). These phagocytes recognize and bind common constitu-

ents of the bacteria, and internalize and destroy them. In addition, the

phagocytes release cytokines and chemokines that recruit and activate

other cells to help control the infection (Sundquist and Wick, 2005;

Kirby et al., 2002; Mastroeni, 2002). However, Salmonella can survive and

replicate within phagocytes, particularly macrophages and DCs (Monack

et al., 2004; Sheppard et al., 2003; Salcedo et al., 2001; Svensson et al.,

2000, 2001, Mariott et al., 1999; Richter-Dahlfors et al., 1997). This

feature makes Salmonella a formidable challenge for the host immune

response.

8.3 PROCESSING OF SALMONELLA FOR DIRECT

PRESENTATION ON MHC-I BY INFECTED DCs

Wild type Salmonella remain confined in vacuolar compartments after

internalization by DCs (Brumell and Grinstein, 2004; Petrovska et al.,

2004; Waterman and Holden, 2003). Once inside DCs, the bacteria are

processed and peptides derived from Salmonella-encoded proteins are

presented on MHC-I (Johannson and Wick, 2004; Yrlid and Wick, 2000,

2002; Niedergang et al., 2000; Svensson et al., 1997, 2000; Svensson

and Wick, 1999). This has been demonstrated using bone marrow-derived

DCs as well as DCs isolated from the spleen, mesenteric lymph nodes or

liver of naive mice infected with Salmonella ex vivo. Moreover, both CD8aþ

and CD8a� splenic DC subsets internalize Salmonella and process the

bacteria for peptide presentation on MHC-I (Yrlid and Wick, 2002).

Direct presentation of Salmonella antigens on MHC-I has been

studied using Salmonella that are internalized by DCs in a process using

actin-driven cytoskeletal rearrangements (Johannson and Wick, 2004;

Yrlid and Wick, 2002; Yrlid et al., 2001). Studies investigating the pathway

used for MHC-I presentation of Salmonella-encoded antigens suggest

that components of the cytosolic MHC-I antigen presentation pathway

are used. This occurs despite the vacuolar localization of the bacteria in

DC (Brumell and Grinstein, 2004; Petrovska et al., 2004; Waterman and

Holden, 2003). Dendritic cells require the transporter associated with

antigen processing (TAP), an ER membrane protein that translocates

peptides from the cytosol into the ER in an ATP-dependent fashion, to

present Salmonella antigens on MHC-I (Yrlid et al., 2001). In addition,

although not formally demonstrated for Salmonella but assessed for

another Gram-negative vacuole-confined bacteria (E. coli), newly synthesized

MHC-I molecules and the proteasome are also required for DC presentation

�161

DE

ND

RIT

ICC

EL

LS,

MA

CR

OP

HA

GE

SA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

�161

DE

ND

RIT

ICC

EL

LS,

MA

CR

OP

HA

GE

SA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

of bacteria-encoded antigens (Svensson and Wick, 1999). It thus appears

that the cytosolic antigen presentation machinery is also used for MHC-I

presentation of bacterial antigens after phagocytic uptake of Salmonella

by DCs.

The mechanism used by Salmonella-infected DCs to present bacterial

antigens on MHC-I appears distinct from that used by macrophages

to present Salmonella antigens. In the case of macrophages, the TAP

transporter and proteasomes are not required (Wick and Ljunggren, 1999;

Song and Harding, 1996; Wick and Pfeifer, 1996). However, more

thorough characterization of the pathway(s) used for MHC-I presentation

of Salmonella antigens by infected DCs, ideally using native bacterial

antigens instead of recombinantly expressed proteins, are needed. In

particular, investigating whether MHC-I presentation of Salmonella

antigens includes a pathway where the endoplasmic reticulum inter-

sects with phagosomes and results in phagosomes capable of TAP-and

proteasome-dependent presentation of bacterial antigens would be informa-

tive (Guermonprez et al., 2003; Houde et al., 2003).

8.4 MODULATING OF ANTIGEN PRESENTATION

BY SALMONELLA

As mentioned above, S. typhimurium has evolved strategies to survive in

phagosomal environments, which otherwise kill phagocytosed bacteria.

It thus follows that the intracellular survival strategies used by Salmonella

may influence the capacity of an infected DC to process and present

bacterial antigens. This, indeed, appears to be the case. For example, the

phoP/phoQ regulatory system, which controls the expression of over

40 genes and is involved in bacterial survival in phagosomal compartments

(Groisman, 2001; Ohl and Miller, 2001), can influence the ability of infected

DCs to present Salmonella-encoded antigens. Antigens from Salmonella

constitutively expressing phoP, such that phoP-activated genes are locked

on and phoP-repressed genes are off, are more efficiently presented on

MHC-II after bacterial internalization by DCs (Svensson et al., 2000).

The effect of phoP/phoQ was apparent when antigen presentation was

quantitated after a short (2 h) but not a longer (24 h) exposure to bacteria

(Niedergang et al., 2000; Svensson et al., 2000). The effect of phoP/phoQ on

antigen presentation by infected DCs required live bacteria, demonstrating

that bacterial gene expression was required for the effect (Svensson et al.,

2000). Thus, the phoP/phoQ regulatory locus can influence the capacity

of DCs to present Salmonella antigens on MHC-II during a short time

�

162

M.

J.W

ICK

frame after bacterial infection. Despite the effect of phoP/phoQ on pre-

sentation of a Salmonella-encoded antigen on MHC-II by infected DCs,

no effect of this locus on presentation of Salmonella antigens on MHC-I

was found (Svensson et al., 2000). The reason underlying the different

effect of phoP/phoQ on MHC-I and MHC-II presentation of Salmonella

antigens has not been established.

A recent report showed that additional virulence-associated genes,

in this case encoded in the Salmonella pathogenicity island 2 (SPI2) region,

reduced the presentation of a Salmonella-encoded antigen on MHC-II by DCs

infected with opsonized bacteria (Cheminay et al., 2005). This was shown in

studies where presentation of an MHC-II epitope of ovalbumin was reduced

when DCs co-cultured with ovalbumin were simultaneously infected with

live, wildtype Salmonella. The reduced MHC-II presentation required viable

bacteria. Furthermore, it was most apparent when DCs were infected with

Salmonella harboring a functional SPI2 locus and when reactive nitrogen

species produced by inducible nitric oxide synthase (iNOS) occurred. The

mechanism of SPI2-mediated interference with MHC-II presentation by

DCs infected with opsonized Salmonella is not fully characterized, but may

have to do with SPI2-mediated modulation of intracellular transport

processes (Cheminay et al., 2005).

In addition to virulence genes, other factors, such as the ease of

bacterial uptake and the amount of antigen present in the bacteria that

are internalized, can influence the efficiency of antigen presentation.

Although the above studies demonstrating a role of phoP/phoQ and

SPI2 in modulating direct presentation of Salmonella antigens by infected

DCs eliminated bacterial uptake and antigen abundance as factors contri-

buting to the observed altered antigen presentation, another study showed

that directing Salmonella to Fcg receptors on DCs by opsonization with

Salmonella-specific IgG enhanced the presentation of a Salmonella-encoded

antigen on MHC-I and MHC-II (Tobar et al., 2004). In contrast to pre-

vious reports (Johannson and Wick, 2004; Yrlid and Wick, 2000, 2002;

Niedergang et al., 2000; Svensson et al., 1997, 2000), Tobar et al. could

only detect presentation of antigens encoded in wild type Salmonella

when the bacteria were opsonized. Based on this they concluded that

virulent Salmonella interferes with the capacity of DCs to process the

bacteria for antigen presentation, and this could be overcome by targeting the

bacteria to Fcg receptors. However, direct evidence for active inhibition

of antigen presentation by Salmonella was lacking. It thus remains possible

that increasing antigen load in the DCs by opsonization, perhaps com-

bined with altered intracellular trafficking when internalized as immune

�163

DE

ND

RIT

ICC

EL

LS,

MA

CR

OP

HA

GE

SA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

complexes, explains the observed effect on antigen presentation. Indeed,

opsonization increases the number of bacteria per DC (Hu et al., 2004;

Eriksson et al., 2003), and targeting antigens to Fcg receptors on DCs

increases antigen presentation in several other settings (Kalergis and

Ravetch, 2002; Guyre et al., 2001; Machy et al., 2000; Regnault et al., 1999).

8.5 DENDRITIC CELLS AS BYSTANDER ANTIGEN

PRESENTING CELLS

The presentation of Salmonella antigens by DCs discussed above focused

on direct antigen presentation by infected cells. In this pathway, DCs

internalize Salmonella, process the bacteria and display MHC molecules

containing bacterial antigens on their cell surface for recognition by T cells.

In other words, the Salmonella-infected DCs directly process the bacteria

and present bacterial antigens to T cells. However, DCs can also present

bacterial antigens when they themselves are not infected by the bacteria

in a process called indirect presentation or, in the case of MHC-I, cross-

presentation. In indirect presentation, the DCs that present Salmonella

antigens are not infected by the bacteria per se. Instead, the DCs are

non-infected bystander cells that acquired Salmonella antigens by inter-

nalizing neighboring cells that have undergone death due to Salmonella

infection. Salmonella expressing the type III secretion system is cytotoxic

to infected cells (Monack et al., 2001), and dead cells cannot productively

interact with T cells (Yrlid and Wick, 2000). However, the indirect

antigen presentation pathway provides a safety valve where DCs ‘‘mop up’’

cell debris containing Salmonella antigens and use this material to

stimulate T cells (Yrlid and Wick, 2000).

Salmonella-induced cell death has been best studied in infected macro-

phages and epithelial cells (Monack et al., 2001). However, Salmonella

can also kill infected DCs by a mechanism dependent on the type III

secretion system (van der Velden et al., 2003; Yrlid et al., 2001). Whether

DCs that have undergone Salmonella-mediated death are also a reservoir

of cell debris containing Salmonella antigens that can be presented by

neighboring, bystander DCs is presently not known. Interestingly, the

capacity to act as a bystander antigen presenting cell appears to be

a unique feature of DCs, as bystander macrophages ingest Salmonella-

induced apoptotic cells but do not present peptides from Salmonella antigens

(Yrlid and Wick, 2000). Data suggest that macrophages compete for apoptotic

material and limit the antigen available for presentation by bystander DCs

(Yrlid and Wick, 2000).

�

164

M.

J.W

ICK

The precise nature of the material in the cell debris responsible for

the observed bystander presentation of Salmonella antigens from apoptotic

macrophages is not known. However, neither peptides released into the

environment that bind preformed surface MHC molecules on bystander

DCs nor bacteria released into the surroundings that are subsequently

phagocytosed and processed by the bystander cells account for the observed

presentation (Yrlid and Wick, 2000). Additional experiments are needed to

characterize the antigenic material derived from the dying, Salmonella-

infected cells.

In the case of Salmonella and other intracellular bacteria, the ability

of DCs to scavenge bacterial antigens from dying cells and present them

to the immune system would allow detection of microbes that may

otherwise be elusive (Winau et al., 2004). Indeed, bystander presentation of

antigenic material from cells induced to undergo apoptosis due to infection

withMycobacterium tuberculosis has also been shown (Schaible et al., 2003). In

the case of this vacuole-confined bacterium, apoptotic macrophages shuttle

vesicles containing mycobacterial lipids and proteins to DCs which in turn

present the material to CD1b- and MHC-I-restricted T cells, respectively

(Schaible et al., 2003). Similarly, Listeria-infected macrophages are a source of

bacterial antigen that can be cross-presented on MHC-I and MHC-II

by bystander DCs (Janda et al., 2004; Skoberne et al., 2002). Unlike

mycobacteria, cross-presentation of Listeria antigens on MHC-I by bystander

DCs did not require apoptotic antigen donor cells, but unstable bacterial

translation products by infected, viable donor cells was instead important

(Janda et al., 2004). Apoptotic macrophages infected with the fungal

pathogen Histoplasma capsulatum are also donors of fungal antigen for

presentation on MHC-I after uptake by bystander DCs (Lin et al., 2005).

Thus, cross-presentation of bacterial and fungal antigens by bystander DCs,

in addition to numerous examples of viral proteins that are cross-presented

by DCs (Fonteneau et al., 2002), has been documented. This supports a role

of indirect presentation of microbial antigens by non-infected DCs as a

means to elicit microbe-specific T cells during infection.

Despite the numerous studies characterizing indirect presentation

of microbial antigens by bystander DCs, relatively little information is

available thus far on cross-presentation of antigens from microbes in vivo

during infection. Recently, however, Salmonella-infected tumor cells

were shown to be a source of tumor antigens that are cross-presented

to CD8þ T cells and contribute to a systemic anti-tumor response in vivo

(Avogadri et al., 2005). Moreover, neutrophils from Listeria-infected mice

are a substrate for bacterial antigens that are cross-presented on MHC-I

�165

DE

ND

RIT

ICC

EL

LS,

MA

CR

OP

HA

GE

SA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

by DCs, particularly non-secreted Listeria antigens that are otherwise

directly presented by neutrophils only very inefficiently (Tvinnereim et al.,

2004). Listeria-infected neutrophils as a source of antigen for cross-priming

was confirmed by showing that neutrophil-enriched cells from infected

mice resulted in cross-priming after adoptive transfer into naive mice, and

by the reduction in CD8þ T cell priming in mice depleted of neutrophils

prior to infection (Tvinnereim et al., 2004).

8.6 CONCLUDING REMARKS

Dendritic cells, which are the pivitol cells in the transition from innate to

adaptive immunity, can either be direct or indirect presenters of Salmonella

antigens. Dendritic cells can also directly and indirectly present antigens

from other pathogens including bacterial, fungal and viral pathogens.

Dendritic cells directly present bacterial antigens to T cells upon phagocytic

processing of bacteria such as Salmonella that does not induce their

death. They can also present bacterial antigens to T cells as bystander

antigen presenting cells that engulf antigenic material from neighboring

cells that have undergone bacteria-induced death. Thus, despite that

Salmonella has evolved mechanisms to (1) survive in phagocytes during

infection, (2) modulate presentation of its antigens by infected DCs and

(3) induce death in infected cells, the exquisite capacity of DCs to directly

and indirectly present Salmonella antigens counters these pathogenic

mechanisms to promote adaptive immunity to this formidable intracellular

pathogen.

REFERENCES

Avogadri, F., Martinoli, C., Petrovska, L. et al. (2005). Cancer immuno-

therapy based on killing of Salmonella-infected tumor cells. Cancer Res.

65, 3920–7.

Banchereau, J. and Steinman, R. M. (1998). Dendritic cells and the control

of immunity. Nature 392, 245–52.

Brumell, J. H. and Grinstein, S. (2004). Salmonella redirects phagosomal

maturation. Curr. Opin. Microbiol. 7, 78–84.

Cheminay, C., Mohlenbrink, A. and Hensel, M. (2005). Intracellular Salmonella

inhibit antigen presentation by dendritic cells. J. Immunol. 174, 2892–9.

Eriksson, S., Chambers, B. J. and Rhen, M. (2003). Nitric oxide produced by

murine dendritic cells is cytotoxic for intracellular Salmonella enterica sv.

Typhimurium. Scand. J. Immunol. 58, 493–502.

�

166

M.

J.W

ICK

Fonteneau, J.-F., Larsson, M. and Bhardwaj, N. (2002). Interactions between dead

cells and dendritic cells in the induction of antiviral CTL responses. Curr.

Opin. Immunol. 14, 471–7.

Groisman, E. A. (2001). The pleiotropic two-component regulatory system

PhoP-PhoQ. J. Bacteriol. 183, 1835–42.

Guermonprez, P., Saveanu, L., Kleijmeer, M., Davoust, J., van Endert, P.,

and Amigorena, S. (2003). ER-phagosome fusion defines an MHC

class I cross-presentation compartment in dendritic cells. Nature 425,

397–402.

Guyre, C. A., Barreda, M. E., Swink, S. L. and Fanger, M. W. (2001). Colocalization

of FcgRI-targeted antigen with class I MHC: implications for antigen

processing. J. Immunol. 166, 2469–78.

Harding, C. V., Ramachandra, L. and Wick, M. J. (2003). Interaction of

bacteria with antigen presenting cells: influences on antigen

presentation and antibacterial immunity. Curr. Opin. Immunol. 15,

112–19.

Houde, M., Bertholet, S., Gagnon, E., Brunet, S., Goyette, G., Laplante, A.

et al. (2003). Phagosomes are competent organelles for antigen

cross-presentation. Nature 425, 402–6.

Hu, P. Q., Tuma-Warrino, R. J., Bryan, M. A., Mitchell, K. G., Higgins, D. E.,

Watkins, S. C. and Salter, R. D. (2004). Escherichia coli expressing

recombinant antigen and listeriolysin O stimulate class I-restricted

CD8þ T cells following uptake by human APC. J. Immunol. 172,

1595–601.

Janda, J., Schoneberger, P., Skoberne, M., Messerle, M., Russmann, H.,

and Geginat, G. (2004). Cross-presentation of Listeria-derived CD8 T cell

epitopes requires unstable bacterial translation products. J. Immunol.

173, 5644–51.

Johannson, C. and Wick, M. J. (2004). Liver dendritic cells present bacterial

antigens and produce cytokines upon Salmonella encounter. J. Immunol.

172, 2496–503.

Jones, B. D., Ghori, N. and Falkow, S. (1994). Salmonella typhimurium

initiates murine infection by penetrating and destroying the specialized

epithelial M cells of the Peyer’s patches. J. Exp. Med. 180, 15–23.

Kalergis, A. M. and Ravetch, J. V. (2002). Inducing tumor immunity through

the selective engagement of activating Fcg receptors on dendritic cells.

J. Exp. Med. 195, 1653–9.

Kirby, A. C., Yrlid, U. and Wick, M. J. (2002). The innate immune response

differs in primary and secondary Salmonella infection. J. Immunol. 169,

4450–9.

�167

DE

ND

RIT

ICC

EL

LS,

MA

CR

OP

HA

GE

SA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

Kirby, A. C., Yrlid, U., Svensson, M. and Wick, M. J. (2001). Differential

involvement of dendritic cell subsets during acute Salmonella infection.

J. Immunol. 166, 6802–11.

Lin, J.-S., Yang, C.-W., Wang, D.-W. and Wu-Hsieh, B. A. (2005). Dendritic

cells cross-present exogenous fungal antigens to stimulate a protective

CD8 T cell response in infection by Histoplasma capsulatum. J. Immunol.

174, 6282–91.

Machy, P., Serre, K. and Leserman, L. (2000). Class I-restricted presentation

of exogenous antigen acquired by Fcg receptor-mediated endocytosis is

regulated in dendritic cells. Eur. J. Immunol. 30, 848–57.

Macpherson, A. J. and Uhr, T. (2004). Induction of protective IgA by intestinal

dendritic cells carrying commensal bacteria. Science 303, 1662–5.

Mariott, I., Hammond, T. G., Thomas, E. K. and Rost, K. L. (1999). Salmonella

efficiently enter and survive within cultured CD11cþ dendritic cells

initiating cytokine expression. Eur. J. Immunol. 29, 1107–15.

Mastroeni, P. (2002). Immunity to systemic Salmonella infections.

Curr. Molec. Med. 2, 393–406.

McSorley, S. J., Asch, S., Costalonga, M., Reinhardt, R. L. and Jenkins, C. (2002).

Tracking Salmonella-specific CD4 T cells in vivo reveals a local mucosal

response to a disseminated infection. Immunity 16, 365–77.

Monack, D. M., Navarre, W. W. and Falkow, S. (2001). Salmonella-induced

macrophage death: the role of caspase-1 in death and inflammation.

Microb. Infect. 3, 1201–12.

Monack, D. M., Bouley, D. M. and Falkow, S. (2004). Salmonella typhimurium

persists within macrophages in the mesenteric lymph nodes of

chronically infected Nramp1þ/þ mice and can be reactivated by IFNg

neutralization. J. Exp. Med. 199, 231–41.

Niedergang, F., Sirard, J.-C., Tallichet Blanc, C. and Kraehenbuhl, J.-P.

(2000). Entry and survival of Salmonella typhimurium in dendritic

cells and presentation of recombinant antigens do not require

macrophage-specific virulence factors. Proc. Natl Acad. Sci. U S A 97,

14650–5.

Niess, J. H., Brand, S., Gu, X., Landsman, L., Jung, S., McCormick, B. A.

et al. (2005). CX3CR1-mediated dendritic cell access to the intestinal lumen

and bacterial clearance. Science 307, 254–8.

Ohl, M. E. and Miller, S. I. (2001). Salmonella: A model for bacterial

pathogenesis. Annu. Rev. Med. 52, 259–74.

Petrovska, L., Aspinall, R. J., Barber, L., Clare, S., Simmons, C. P., Stratford, R.

et al. (2004). Salmonella enterica serovar Typhimurium interaction

with dendritic cells: impact of the sifA gene. Cellular Microbiol. 6, 1071–84.

�

168

M.

J.W

ICK

Regnault, A., Lankar, D., Lacabanne, V., Rodriguez, A., Thery, C., Rescigno, M.

et al. (1999). Fcg receptor-mediated induction of dendritic cell maturation

and major histocompatibility complex class I-restricted antigen presentation

after immune complex internalization. J. Exp. Med. 189, 371–80.

Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R.

et al. (2001). Dendritic cells express tight junction proteins and

penetrate gut epithelia monolayers to sample bacteria. Nature Immunol.

2, 361–7.

Richter-Dahlfors, A., Buchan, A. M. J. and Finlay, B. B. (1997). Murine

salmonellosis studied by confocal microscopy: Salmonella typhimurium

resides intracellularly inside macrophages and exerts a cytotoxic effect

on phagocytes in vivo. J. Exp. Med. 186, 569–80.

Rock, K. L. and Goldberg, A. L. (1999). Degradation of cell proteins and

the generation of MHC class I-presented peptides. Annu. Rev. Immunol.

17, 739–79.

Salcedo, S. P., Noursadeghi, M., Cohen, J. and Holden, D. W. (2001). Intracellular

replication of Salmonella typhimurium strains in specific subsets of splenic

macrophages in vivo. Cell. Microbiol. 3, 587–97.

Schaible, U. E., Winau, F., Sieling, P. A., Fischer, K., Collins, H. L., Hagens, K.

et al. (2003). Apoptosis facilitates antigen presentation to T lymphocytes

through MHC-I and CD1 in tuberculosis. Nature Med. 9, 1039–46.

Sheppard, M., Webb, C., Heath, F., Mallos, V., Emilianus, R., Maskell, D.,

and Mastroeni, P. (2003). Dynamics of bacterial growth and

distribution within the liver during Salmonella infection. Cellular Microbiol.

5, 593–600.

Skoberne, M., Schenk, S., Hof, H. and Geginat, G. (2002). Cross-presentation

of Listeria monocytogenes-derived CD4 T cell epitopes. J. Immunol. 169,

1410–18.

Song, R. and Harding, C. V. (1996). Roles of proteasomes, transporter for antigen

presentation (TAP), and b2-microglobulin in the processing of bacterial or

particulate antigens via an alternate class I MHC processing pathway.

J. Immunol. 156, 4182–90.

Sundquist, M. and Wick, M. J. (2005). TNF-a-dependent and-independent

maturation of dendritic cells and recruited CD11cintCD11bþ cells during

oral Salmonella infection. J. Immunol. 175, 3287–98.

Sundquist, M., Rydstrom, A. and Wick, M. J. (2004). Immunity to Salmonella

from a dendritic point of view. Cell. Microbiol. 6, 1–11.

Svensson, M. and Wick, M. J. (1999). Classical MHC-I presentation of a

bacterial fusion protein by bone marrow-derived dendritic cells.

Eur. J. Immunol. 29, 180–8.

�169

DE

ND

RIT

ICC

EL

LS,

MA

CR

OP

HA

GE

SA

ND

CR

OS

S-PR

ES

EN

TA

TIO

NO

FB

AC

TE

RIA

LA

NT

IGE

NS

Svensson, M., Stockinger, B. and Wick, M. J. (1997). Bone marrow-derived

dendritic cells can process bacteria for MHC-I and MHC-II presentation

to T cells. J. Immunol. 158, 4229–36.

Svensson, M., Johansson, C. and Wick, M. J. (2000). Salmonella enterica

serovar Typhimurium-induced maturation of bone marrow-derived

dendritic cells. Infect. Immun. 68, 6311–20.

Svensson, M., Johannson, C. and Wick, M. J. (2001). Salmonella

typhimurium-induced cytokine production and surface molecule

expression by murine macrophages. Microb. Pathog. 31, 91–102.

Tobar, J. A., Gonzalez, P. A. and Kalergis, A. M. (2004). Salmonella

escape from antigen presentation can be overcome by targeting

bacteria to Fcg receptors on dendritic cells. J. Immunol. 173, 4058–65.

Tvinnereim, A. R., Hamilton, S. E. and Harty, J. T. (2004). Neutrophil

involvement in cross-priming CD8þ T cell responses to bacterial

antigens. J. Immunol. 173, 1994–2002.

van der Velden, A. W. M., Velassquez, M. and Starnbach, M. N. (2003). Salmonella

rapidly kill dendritic cells via a caspase-1-dependent mechanism.

J. Immunol. 171, 6742–9.

Waterman, S. R. and Holden, D. W. (2003). Functions and effectors of

the Salmonella pathogenicity island 2 type III secretion system. Cellular

Microbiol. 5, 501–11.

Wick, M. J. and Pfeifer, J. D. (1996). MHC-I presentation of OVA(257–264) from

exogenous sources: protein context influences the degree of

TAP-independent presentation. Eur. J. Immunol. 26, 2790–9.

Wick, M. J. and Ljunggren, H.-G. (1999). Processing of bacterial antigens

for peptide presentation on MHC class I molecules. Immunol. Rev. 172,

153–62.

Winau, F., Kaufmann, S. H. E. and Schaible, U. E. (2004). Apoptosis paves

the detour path for CD8 T cell activation against intracellular bacteria.

Cellular Microbiol. 6, 599–607.

Yrlid, U. and Wick, M. J. (2000). Salmonella-induced apoptosis of

infected macrophages results in presentation of a bacteria-encoded

antigen after uptake by bystander dendritic cells. J. Exp. Med. 191,

613–23.

Yrlid, U. and Wick, M. J. (2002). Antigen presentation capacity and cytokine

production by murine splenic dendritic cell subsets upon Salmonella

encounter. J. Immunol. 169, 108–16.

Yrlid, U., Svensson, M., Kirby, A. C. and Wick, M. J. (2001). Antigen-presenting

cells and anti-Salmonella immunity. Microb. Infect. 3, 1239–48.

�

170

M.

J.W

ICK

PART IV Dendritic cells and immuneevasion of bacteria in vivo

CHAPTER 9

Pathogen-recognition receptors as targetsfor pathogens to modulate immune functionof antigen-presenting cells

Anneke Engering, Sandra J. van Vliet, Estella A. Koppel, Teunis

B. H. Geijtenbeek and Yvette van KooykVU Medical Center

9.1 INTRODUCTION

Antigen-presenting cells (APC), such as dendritic cells (DCs) and macro-

phages, are located throughout the body to sense and capture invading

pathogens and to trigger immune responses to fight such invaders.

In addition, in the absence of danger signals, DCs have an active role in

the induction of T cell tolerance and the maintenance of homeostasis.

The recognition and internalization of pathogens is mediated by so-called

pathogen-recognition receptors, germ-line encoded cell surface receptors

that include toll-like receptors (TLR) and C-type lectins (CLR). It is becoming

increasingly clear that during the long co-evolution with their hosts,

pathogens have evolved mechanisms to misuse pathogen-recognition

receptors to suppress or evade immune responses and thus to escape

clearance. In this chapter, we will review recent examples of how pathogens

evade immune activation by targeting recognition receptors on APC and

subverting their function.

9.2 BACTERIAL RECEPTORS ON ANTIGEN-PRESENTING CELLS

APC interact with invading pathogens via pathogen-recognition receptors

that bind conserved patterns of carbohydrates, lipids, proteins and nucleic

acids in classes of microbes1,2. This variety of receptors and conserved

ligands recognized ensures that most, if not all, microbes can be detected

by the immune system, either by a single or by combinations of receptors.

Pathogen-recognition receptors include TLR3 and CLR4 (Figure 9.1).

To date, 11 TLR have been identified (see Chapter 2) that each targets

specific pathogenic structures, such as lipopolysaccharide (TLR4), viral

dsRNA (TLR3) and bacterial peptidoglycans (TLR2/TLR6)5. Upon inter-

action with a pathogen, TLR transmit this information through signalling

pathways resulting in activation of APC, including expression of costimu-

latory molecules and production of inflammatory cytokines (Figure 9.1).

APC react differently to distinct microbial ligands, even though there is

an overlap of adaptor molecules and signalling routes between different

TLR. Indeed, recent studies support the hypothesis that different TLR

ligands activate distinct downstream responses leading to tailored activation

of APC to most effectively fight the specific pathogen6.

Whereas TLR are instrumental for alerting the immune system for

invading pathogens, other pathogen receptors on APC play a role in

capturing the microbes for intracellular degradation and antigen presenta-

tion. These include the family of CLR that recognize pathogens by their

carbohydrate structures7. However, the range of ligands that interact with

CLR include not only pathogenic structures but also self molecules,

mediating cell�cell adhesion as well as internalizing endogenous ligands

Figure 9.1. TLR and CLR are pathogen-recognition receptors on APC. APC express

several classes of receptors that mediate recognition and internalization of pathogens,

including TLR and CLR. Recognition of pathogens or their components by TLR leads

to activation of intracellular signalling pathways, including NFkB translocation,

and subsequent upregulation of expression of costimulatory and adhesion molecules

and production of cytokines. CLR bind carbohydrate structures on pathogens resulting

in internalization, and intracellular processing for presentation by MHC II molecules

to T cells. TCR, T cell receptor.

�

174

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

for homeostatic control8. Binding to carbohydrates is mediated by the

carbohydrate-recognition domain (CRD) of the CLR9. The more than 15 CLR

that have been cloned from DC and macrophages so far either contain

a single CRD, including DC-SIGN, MGL, and Dectin-1, or multiple CRDs,

such as MR (8 CRDs) and DEC-205 (10 CRDs). The exact carbohydrate

moieties recognized by the distinct CLR are currently being explored

and depend not only on the type of glycan (such as mannose, fucose or

galactose) but also on the complexity, multivalency, and branching of the

carbohydrates (see http://web.mit.edu/glycomics/consortium/).

Most CLR are highly expressed on immature DCs and function as

endocytic receptors that capture antigens for presentation (Figure 9.1).

Distinct internalization motifs present in the cytoplasmic part of several

CLR can mediate and guide endocytosis and intracellular trafficking4.

For example, ligand binding to DC-SIGN triggers internalization into

lysosomal compartments, whereas MR continuously recycles between the

cell surface and early endosomes, where ligand is released10�12. Overall,

such receptor-mediated antigen uptake results in high amounts of

internalized antigens for antigen processing and presentation to T cells.

However, several pathogens including HIV-1 have evolved to subvert these

internalization routes resulting in poor antigen-presentation (see below).

In addition to endocytic motifs, some CLR contain putative signalling

motifs including immunoreceptor tyrosine-based inhibitory (ITIM) and

activatory (ITAM) motifs, indicating that such CLR have immuno-

suppressive or activatory functions4.

Other families of pathogen-recognition receptors include the cyto-

plasmic surveillance proteins NOD1 and NOD2, scavenger receptors, other

lectin receptors (e.g. galectins) and opsonic receptors for immunoglobulins

(FcR) and complement1,13. Each of these receptors can potentially trigger

intracellular signals and thus contribute to the final outcome of the immune

response. On the other hand, during co-evolution of pathogens with their

hosts, each of these receptors could have provided means for pathogens

to modulate, or even evade, immune responses.

9.3 PATHOGENS ARE RECOGNIZED BY COMBINATIONS

OF RECEPTORS

Although many studies have focused on isolated ligands for pathogen-

recognition receptors, in the body APC will mainly encounter intact

pathogens that contain a variety of potential recognition elements.

In addition, during destruction of pathogens upon internalization into the

�175

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

endosomal/lysosomal pathway, alternative ligands can become available

for triggering of intracellularly expressed receptors. TLR9, for instance,

is localized intracellularly in endosomes and recognizes DNA derived

from internalized and degraded bacteria14. Thus, whole microbes will be

recognized by combinations of distinct receptors both on the cell surface

and intracellular. Simultaneous and/or sequential activation of multiple

innate receptors will assist in tailoring an effective response to a specific

pathogen. Besides intact pathogens that are recognized during initial

infection, at later stages and during chronic infection soluble microbial

components can be secreted that bind to recognition receptors on APC.

For example Mycobacterium tuberculosis infects primarily macrophages,

but at later stages after infection induces secretion of its cell wall

component mannosylated lipoarabinomannan (ManLAM), that modulates

DC function (see below).

Moreover, different APC express distinct combinations of pathogen-

recognition receptors; both the amounts and the relative abundance

can vary. For example, whereas DCs express the CLR DC-SIGN and MR

and low amounts of TLR1 and TLR2, monocytes/macrophages express

MR, but not DC-SIGN, and high levels of TLR1 and TLR215,16. This will

result in distinct immune effector responses depending on the subset

of APC that encounters a pathogen. Also, the availability and the affinity

of the receptors will determine whether they are triggered or not.

Selective expression of pathogen-recognition receptors in defined intracel-

lular compartments together with the route of the pathogen upon

internalization will govern ligand interactions. In addition, receptors can

have overlapping ligand specificity but may differ in affinity for pathogenic

component, thus resulting in dominance of one receptor for interacting

with a specific pathogen. Detailed knowledge on expression, ligand

specificity and affinity of recognition receptors as well as on their signall-

ing capacities is required to decipher the immune response triggered by

an intact pathogen.

9.4 BACTERIA CAN USE TLR TO EVADE IMMUNE RESPONSES

TLR play a crucial role in alarming the immune system upon invasion

of pathogens3. Upon ligation of TLR, DCs are activated and migrate from

the sites of infection to the lymph nodes, for antigen presentation to T cells.

Thus, DCs bridge innate and acquired immune responses. The importance

�

176

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

of TLR became clear in mice and humans that lack specific TLR

or intracellular signalling components, leading to an increased susceptibility

to a variety of microbes. In addition, such models have identified specific

ligands for each TLR; for example products from Gram-positive bacteria are

recognized by TLR2, whereas TLR4 mediates recognition of Gram-negative

bacteria and their components. However, besides activation of efficient

immune responses, recent findings show that certain pathogens misuse

TLR to escape the host defense17.

Several bacteria have found a way to control inflammation by modifica-

tion of TLR ligands. In general, TLR recognize conserved microbial

compounds that are essential for pathogenicity and survival of pathogens.

However, it is becoming clear that modifications of TLR ligands do

occur between species and even within one species, thereby altering the

interaction and subsequent signalling of TLR. One example is lipid A,

the part of lipopolysaccharide that is recognized by TLR4 and that is an

essential component of the outer membrane of Gram-negative bacteria.

Whereas E. coli-derived lipid A is a strong activator of TLR4, lipid A

from other bacteria can be modified resulting in low affinity (e.g. LPS from

Salmonella18) or even escape from recognition by TLR4 (e.g. LPS from

P. gingivalis19). In addition to modification of TLR ligands, pathogens can

block specific components of the TLR signalling pathway, thereby reducing

activation of APC and immune responses.

Other bacteria have found means to directly induce immune sup-

pression instead of immune activation by signalling through TLR2. In

comparison to TLR4 activation, TLR2 ligation induces weak inflam-

matory responses and strong anti-inflammatory effects20. This is probably

part of the recovery phase that occurs after inflammation and clearance

to allow the body to return to normal steady-state conditions. Recent

studies have shown that pathogens exploit this anti-inflammatory effect of

TLR2 to avoid inflammation and thus promote survival20 (Figure 9.2b).

Examples include pathogenic components that solely trigger TLR2, such

as phosphatidyl serine derived from Schistosoma that activate DCs to

induce regulatory T cells21, or bacteria that reduce TLR4 triggering in

favor of TLR2 activation like P. gingivalis19 and Yersinia that induces

immunosuppression via IL-1022. However, it is not clear if TLR2 is the

only receptor that is involved in the described immunosuppression or

that members of other pathogen receptor families are simultaneously

triggered, such as CLR (see below).

�177

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

Figure 9.2. Pathogens are recognized by combinations of receptors, but can also

target these receptors to escape immune responses. (a) Self-antigens that bind to CLR

in steady-state conditions can be internalized by APC, but no activated occurs resulting

in induction of tolerance. However, upon simultaneous recognition of pathogens by CLR

and TLR, APC become activated and effective immune responses are induced. (b) Certain

pathogens, such as P. gingivalis, can modify its TLR ligands to induce immune suppression

by reducing TLR4 triggering in favor of TLR2 activation. (c) Cross-talk between TLR and

CLR can either result in immune suppression or in synergistic activation of APC. Certain

ligands of the MR and DC-SIGN induce down modulation of TLR signalling and inhibition

of immune activation, whereas collaborative signalling of Dectin-1 and TLR2 upon binding

of yeast induces enhanced activation of APC.

�

178

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

9.5 TARGETING TO CLR CAN INDUCE EITHER TOLERANCE

OR IMMUNE ACTIVATION

In contrast to TLR, many CLR were initially identified as receptors for

endogenous (‘‘self’’) ligands. The MR plays a role in the removal of self

glycoproteins from the circulation, whereas MR, DC-SIGN and Dectin-1

are involved in cell�cell interactions23�27. In addition, triggering of CLR

in the absence of inflammatory stimuli does not lead to immune activation

but rather to unresponsiveness. This was first shown by targeting antigens

to DEC-205 on immature DCs, resulting in tolerance and the deletion

of effector T cells28. This leads to the hypothesis that the physiological

function of these receptors is the recognition of glycosylated self-antigens

for homeostatic control and that pathogens have misused CLR to escape

clearance (Figure 9.2a). However, recent evidence indicates that some

CLR, such as Dectin-1, function as a pathogen receptor that activates

APC. Several studies of pathogens that simultaneously trigger CLR and

TLR will be discussed below, showing that cross-talk between CLR and

TLR can either lead to suppression or to activation of immune responses

(Figure 9.2c).

9.6 CROSS-TALK BETWEEN CLR AND TLR CAN RESULT

IN IMMUNE SUPPRESSION

A recent study shows that targeting MR with an activating anti-MR anti-

body or with certain natural ligands could prime a regulatory program in

DCs, leading to the production of anti-inflammatory cytokines and induction

of Th2 cells with regulatory capacity29. MR triggering could also prevent the

production of inflammatory cytokines by the TLR4-ligand LPS implying

cross-talk between the MR and TLR4 leading to down modulation of TLR4

signalling (Figure 9.2c). Interestingly, only a restricted set of ligands, such as

mycobacterial-derived ManLAM and a complex proteoglycan could prime

regulatory DCs, whereas other ligands including mannan and dextran,

had no effect on DCs29. This differential effect could be due to the fact

that several of these ligands can also bind to other C-type lectins, including

DC-SIGN, and might thus influence activation of DCs.

DC-SIGN is a DC-specific CLR that binds self-glycoproteins as well as

a broad range of pathogens through high mannose and Lewis-containing

carbohydrates (Figure 9.3). Although DC-SIGN has been shown to mediate

internalization of ligands for antigen presentation10, certain pathogens,

such as HIV and HCV, induce misrouting after internalization through

�179

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

DC-SIGN to non-lysosomal compartments, allowing escape from degrada-

tion and antigen presentation30,31. In this way, a part of the internalized

HIV remains virulent and can infect T cells in trans upon migration of DCs

to lymph nodes32,33. In addition to HIV, DC-SIGN binds and internalizes

Dengue virus, HCMV, HCV and Ebola for in-trans infection of target

cells34�36 (Figure 9.3).

Figure 9.3. Pathogens target DC-SIGN to escape immune responses. Immature DC

express CLR such as DC-SIGN that recognizes different pathogens through their

carbohydrate structures. The immunological outcome of this interaction is specific for the

pathogen and also depends on co-ligation of other pathogen receptors, such as TLR. Viruses

can use DC-SIGN for transmission to T cells, whereas several bacteria can modulate DC

function through DC-SIGN.

�

180

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

In addition to viruses, other pathogens also target DC-SIGN, including

mycobacteria, yeast and schistosoma parasites37�39. Recognition of such

whole pathogens relies not only on DC-SIGN, but also on other pathogen

receptors. We will highlight three examples of bacteria that simultaneously

trigger DC-SIGN and other pathogen-recognition receptors to modulate

DC function (Figure 9.2c and Figure 9.3).

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the

major infectious diseases worldwide. Although M. tuberculosis primarily

infects macrophages, it also binds DC-SIGN on DCs through interactions

with ManLAM, a major component of its cell wall that can be secreted

by infected cells40,41. Recent studies have demonstrated that ManLAM

binding to DC-SIGN blocks TLR4-induced maturation of DCs and

induces production of the anti-inflammatory cytokine IL-1040. This indicates

that binding of ManLAM to DC-SIGN triggers inhibitory signals that

may enable pathogens such as M. tuberculosis to suppress immune activa-

tion signals through TLR.

The human gastric pathogen Helicobacter pylori causes persistent

infection in half of mankind, which requires a certain balance between

mild inflammation and escape of clearance. H. pylori has evolved many

complex mechanisms to subvert innate and adaptive immunity in its

host, such as resistance to phagocytic killing, inhibition of antigen process-

ing and suppression of T cell activation42. Recently, we have shown that

differential targeting of DC-SIGN on DCs provides H. pylori with yet

another mechanism to modulate immune responses43. H. pylori LPS

express Lewis antigens that are subject to phase variation, meaning that

the genes responsible for these epitopes are switched on and off with

high frequency44. Lewis phase variation is caused by frame shifts in

glycosyltransferase genes and results in LPS with and without Lewis

carbohydrates on bacteria within a single strain. Although both variants

trigger TLR2 and TLR4 to a similar extent, these modifications result in

a differential activation to DCs through targeting of DC-SIGN43. H. pylori

that express Lewis-containing LPS bind to DC-SIGN and inhibit

Th1-polarization compared to Lewis-negative variants that are not recog-

nized by DC-SIGN. The Lewis-negative and -positive strains are iden-

tical except for the presence of Lewis carbohydrates on LPS, implying

that H. pylori uses phase variation to regulate targeting to DC-SIGN and to

modulate DC function. In addition to H. pylori other pathogens that target

DC-SIGN are also likely to block Th1 skewing, such as Schistosoma mansoni

and Leishmania species that both favor Th2, and not Th1, responses for

chronic infections.

�181

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

Besides these Th1/Th2 modulatory effects of DC-SIGN, we have

found that DC-SIGN targeting by Lactobacillus can prime mature

regulatory DCs that induce regulatory T cell differentiation45. Lactobacilli

are one of the most frequently used probiotics, i.e. live microbial food

ingredients that are beneficial to health. Whereas several lactobacilli bound

to DCs, only L. reuteri and L. casei that target DC-SIGN could prime the

development of regulatory T cells. Anti-DC-SIGN antibodies abrogated

this suppressor activity, indicating a crucial role for DC-SIGN herein.

Interestingly, addition of maturation stimuli was needed for full regulatory

T cell-inducing capacity of DCs by lactobacilli, suggesting that cross-

regulation between DC-SIGN and other signalling pathways occurs.

Interestingly, mSIGNR1, the murine homologue of DC-SIGN, is not

expressed on DCs but on subsets of macrophages in lymph nodes, spleen

and liver at locations important in the defence against pathogens46. Several

pathogens have been identified to interact with mSIGNR1 in vitro, such

as the virus HIV-147, the yeast C. albicans48, the mycobacteria M. tuberculosis

and M. bovis BCG40,48 as well as bacteria including Streptococcus pneumoniae,

E. coli and S. typhimurium49,50. The importance of mSIGNR1 during

infection was demonstrated using mice deficient for mSIGNR1; such

mice are more susceptible to S. pneumoniae challenge51. In contrast to

DC-SIGN, that suppresses or modulates TLR signalling, mSIGNR1 was

recently demonstrated to enhance TLR4-mediated cytokine production

by macrophages upon binding to core polysaccharides of LPS52. mSIGNR1

lacks signalling motifs in its cytoplasmic tail, but its association with TLR4

might be involved in the observed synergistic effect52. The generation

of knockout and double-knockout mice will enable further insight in the

function of mSIGNR1 as well as cross-talk with TLR.

9.7 CROSS-TALK BETWEEN CLR AND TLR CAN RESULT

IN IMMUNE ACTIVATION

Another example of cross-talk between CLR and TLR signalling was shown

recently for Dectin-1 and TLR2 that synergize to activate inflammatory

responses. Dectin-1 is a CLR with a single CRD that binds b-glucans,

such as present in yeast, and is expressed by DCs and macrophages53. In

its cytoplasmic tail, Dectin-1 contains an activatory tyrosine-based motif

(ITAM); ligand binding induces tyrosine phosphorylation. In addition to

Dectin-1, yeast particles also trigger signalling through TLR2/6 hetero-

dimers resulting in an inflammatory response54. Two recent papers have

shown that upon simultaneous recognition of heat-killed yeast by Dectin-1

�

182

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

and TLR2/6 collaborative signalling of these two pathogen receptors

induces enhanced inflammatory cytokine production by APC55,56. Dectin-1

is not only involved in binding of yeast through b-glucans, it also mediates

phagocytosis, whereas TLR are not involved in this process54. Strikingly,

the signalling pathway of Dectin-1 differs from the TLR pathway, but

also from other phagocytic receptors such as FcR57, and involves phos-

phorylation of the cytoplasmic tyrosine and recruitment of Syk kinase58.

Interestingly, besides the CLR Dectin-1, several other CLR can also

bind yeast, including MR and DC-SIGN37,39, although the glycans that

are recognized differ. DC-SIGN and the MR bind yeast mannan, whereas

Dectin-1 interacts with b-glucans. Moreover, unicellular yeast is differ-

entially recognized from the virulent filamentous form (hyphae) of Candida

by DC. b-Glucans, which are recognized by Dectin-1, are normally shielded

on live yeasts by the outer wall components. The normal mechanism of yeast

budding and cell separation creates scars that expose enough b-glucans to

trigger Dectin-1 mediated phagocytosis. During filamentous growth no cell

separation and therefore no scarring occurs and the pathogen fails to activate

Dectin-159. Other studies have shown that DCs induce protective Th1

responses upon unicellular yeast phagocytosis but pathology and Th2

induction after hyphae uptake, although the contribution of the different

receptors in such differentiation remains unclear60,61. To fully understand

the immune response triggered by whole yeast, the complex interplay

between multiple receptors involved in recognition has to be further

unraveled. Comparison of gene expression profiles of DCs in response to

Candida albicans or isolated yeast mannan revealed that although a common

gene program is induced by both compounds, mannan activated an

additional set of genes62. This implies that recognition is indeed different

between whole yeast and its cell-wall component mannan and results in

differential activation of DCs.

9.8 OTHER EXAMPLES OF CROSS-TALK BETWEEN

PATHOGEN RECEPTORS

The complexity of recognition of pathogens and their components was

emphasized in a recent study on outer membrane protein A (OmpA) of

Klebsiella63. Whereas recognition by macrophages and DCs is mediated

by the scavenger receptors Lox-1 and SREC-1, TLR2 triggering results in

activation, including production of the soluble pathogen-recognition recep-

tor PTX3. Subsequently, PTX3 binds OmpA and enhances inflammatory

responses63. Immune responses to whole Klebsiella will probably be the

�183

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

result of triggering not only Lox-1, SREC-1, TLR-2 and PTX3 that recognize

OmpA, but additional receptors recognizing other Klebsiella components that

may function complementary or contradictory.

In macrophages, synergy was reported between TLR 2, 4, 7 and 9 and

the receptor for adenosine, a product of tissue damage/inflammation.

Concomitant stimulation results in increased production of vascular

endothelial growth factor and downregulation of TNF-a64. Such synergy

could play a role in tissue repair and resolving inflammation after

clearance of the pathogen.

Depending on the stimuli received macrophages can adopt different

modes of action65,66. Classically activated macrophages develop after IFNgactivation along with exposure to a microbe or a microbial product such

as LPS. These cells are easily identified via their production of high levels

of nitric oxide (NO) and reactive oxygen species (ROS). Classically acti-

vated macrophages produce high amounts of IL-12 and stimulate Th1

responses. They also possess an enhanced ability to kill intracellular

pathogens. If however TLR activation is accompanied by FcR triggering via

immune complexes, macrophages differentiate into type II, alternatively

activated macrophages. These macrophages no longer produce IL-12;

instead they secrete high amounts of IL-10. By virtue of this IL-10 production,

type II-activated macrophages have a strong anti-inflammatory function

and preferentially induce Th2-type responses. Upon parasitic infections,

expression of the galactose-type CLR MGL2 is induced on murine alter-

natively activated macrophages67. Interestingly, mMGL2 is highly homo-

logous to human MGL68 that was recently shown to bind glycan structures

containing terminal GalNAc moieties, expressed among others by the

human helminth parasite Schistosoma mansoni69, pointing to a role for

MGL as a pathogen receptor.

9.9 CONCLUDING REMARKS

As discussed above, recent advances indicate that the balance between

pathogen-recognition receptors upon encountering of a pathogen by APC

will determine the outcome of an immune response, either immune

activation or suppression. In order to fully understand the impact of

pathogen recognition, not only the ligand specificity and subsequent

signalling pathway needs to be elucidated, but also studies on highly defined

pathogenic components and/or molecularly modified pathogens, on differ-

ent DCs and macrophage subsets and in animal models are required.

�

184

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

On the other hand, information on the pathogen itself and variations are

crucial to be able to comprehend immune responses.

For several CLR, the outcome of the subsequent immune response

against a pathogen depends on the context in which the ligand is recognized

(i.e. activation of APC through other receptors). In case of self-glycoproteins

that bind to CLR but do not activate APC, immune tolerance will be

generated, whereas pathogens that bind to CLR and strongly activate

TLR will induce antigen presentation to T cells by fully activated DCs

and generation of protective immune responses. However, pathogens

that down modulate TLR activation or provide a dominant signal through

specific CLR, such as DC-SIGN and/or MR, can inhibit immune activa-

tion to promote survival. Such information could be used to suppress

unwanted inflammatory responses as in autoimmunity, for example by

targeting antigens to specific CLR on DCs without additional triggers

for tolerance induction or to TLR2 to induce anti-inflammatory processes.

Likewise, lessons could be learned from triggering combinations of

pathogen receptors that result in synergistic inflammatory effects, in order

to enhance immune responses to tumor antigens or for use in vaccine

development.

REFERENCES

1. Taylor, P. R., L. Martinez-Pomares, M. Stacey, H. H. Lin, G. D. Brown, and

S. Gordon (2005). Macrophage receptors and immune recognition. Annu.

Rev. Immunol. 23, 901�44.

2. Sousa, Reis E. (2004). Activation of dendritic cells: translating innate

into adaptive immunity. Curr. Opin. Immunol. 16, 21�5.

3. Akira, S., K. Takeda, and T. Kaisho (2001). Toll-like receptors: critical

proteins linking innate and acquired immunity. Nat. Immunol. 2, 675�80.

4. Figdor, C. G., Y. van Kooyk, and G. J. Adema (2002). C-type lectin receptors

on dendritic cells and Langerhans cells. Nature Rev. Immunol. 2, 77�84.

5. Underhill, D. M. and A. Ozinsky (2002). Toll-like receptors: key mediators

of microbe detection. Curr. Opin. Immunol. 14, 103�10.

6. McGettrick, A. F. and L. A. O’Neill (2004). The expanding family of MyD88-

like adaptors in Toll-like receptor signal transduction. Mol. Immunol. 41,

577�82.

7. van Kooyk, Y., A. Engering, A. N. Lekkerkerker, I. S. Ludwig, and

T. B. Geijtenbeek (2004). Pathogens use carbohydrates to escape

immunity induced by dendritic cells. Curr. Opin. Immunol. 16, 488�93.

�185

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

8. Geijtenbeek, T. B., S. J. van Vliet, A. Engering, B. A. ’t Hart, and

Y. van Kooyk (2004). Self- and nonself-recognition by C-type lectins on

dendritic cells. Annu. Rev. Immunol. 22, 33�54.

9. Drickamer, K. (1999). C-type lectin-like domains. Curr. Opin. Struct. Biol.

9, 585�90.

10. Engering, A., T. B. Geijtenbeek, S. J. van Vliet, M. Wijers, E. van Liempt,

N. Demaurex, A. Lanzavecchia, J. Fransen, C. G. Figdor, V. Piguet, and

Y. van Kooyk (2002). The dendritic cell-specific adhesion receptor

DC-SIGN internalizes antigen for presentation to T cells. J. Immunol.

168, 2118�26.

11. Tan, M. C., A. M. Mommaas, J. W. Drijfhout, R. Jordens, J. J. Onderwater,

D. Verwoerd, A. A. Mulder, A. N. van der Heiden, D. Scheidegger,

L. C. Oomen, T. H. Ottenhoff, A. Tulp, J. J. Neefjes, and F. Koning (1997).

Mannose receptor-mediated uptake of antigens strongly enhances HLA

class II-restricted antigen presentation by cultured dendritic cells.

Eur. J. Immunol. 27, 2426�35.

12. Engering, A. J., M. Cella, D. Fluitsma, M. Brockhaus, E. C. Hoefsmit,

A. Lanzavecchia, and J. Pieters (1997). The mannose receptor functions

as a high capacity and broad specificity antigen receptor in human

dendritic cells. Eur. J. Immunol. 27, 2417�25.

13. Inohara, N., M. Chamaillard, C. McDonald, and G. Nunez (2005).

NOD-LRR proteins: role in host�microbial interactions and

inflammatory disease. Annu. Rev. Biochem. 74, 355�83.

14. Wagner, H. (2004). The immunobiology of the TLR9 subfamily. Trends

Immunol. 25, 381�6.

15. McGreal, E. P., J. L. Miller, and S. Gordon (2005). Ligand recognition

by antigen-presenting cell C-type lectin receptors. Curr. Opin. Immunol.

17, 18�24.

16. Kokkinopoulos, I., W. J. Jordan, and M. A. Ritter (2005). Toll-like receptor

mRNA expression patterns in human dendritic cells and monocytes.

Mol. Immunol. 42, 957�68.

17. Portnoy, D. A. (2005). Manipulation of innate immunity by bacterial

pathogens. Curr. Opin. Immunol. 17, 25�8.

18. Kawasaki, K., R. K. Ernst, and S. I. Miller (2004). 3-O-deacylation of

lipid A by PagL, a PhoP/PhoQ-regulated deacylase of Salmonella

typhimurium, modulates signaling through Toll-like receptor 4. J. Biol.

Chem. 279, 20044�8.

19. Darveau, R. P., T. T. Pham, K. Lemley, R. A. Reife, B. W. Bainbridge,

S. R. Coats, W. N. Howald, S. S. Way, and A. M. Hajjar (2004).

Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A

�

186

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

species that functionally interact with both Toll-like receptors 2 and 4.

Infect. Immun. 72, 5041�51.

20. Netea, M. G., J. W. Van Der Meer, and B. J. Kullberg (2004). Toll-like

receptors as an escape mechanism from the host defense. Trends

Microbiol. 12, 484�8.

21. van der Kleij, D., E. Latz, J. F. Brouwers, Y. C. Kruize, M. Schmitz,

E. A. Kurt-Jones, T. Espevik, E. C. de Jong, M. L. Kapsenberg,

D. T. Golenbock, A. G. Tielens, and M. Yazdanbakhsh (2002). A novel

host-parasite lipid cross-talk. Schistosomal lyso-phosphatidylserine

activates toll-like receptor 2 and affects immune polarization. J. Biol.

Chem. 277, 48122�9.

22. Sing, A., D. Rost, N. Tvardovskaia, A. Roggenkamp, A. Wiedemann,

C. J. Kirschning, M. Aepfelbacher, and J. Heesemann (2002). Yersinia

V-antigen exploits toll-like receptor 2 and CD14 for interleukin

10-mediated immunosuppression. J. Exp. Med. 196, 1017�24.

23. Geijtenbeek, T. B. H., R. Torensma, S. J. van Vliet, G. C. F. van Duijnhoven,

G. J. Adema, Y. van Kooyk, and C. G. Figdor (2000). Identification of

DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that

supports primary immune responses. Cell 100, 575�85.

24. Geijtenbeek, T. B., D. J. Krooshoop, D. A. Bleijs, S. J. van Vliet,

G. C. van Duijnhoven, V. Grabovsky, R. Alon, C. G. Figdor, and

Y. van Kooyk (2000). DC-SIGN-ICAM-2 interaction mediates dendritic

cell trafficking. Nat. Immunol. 1, 353�7.

25. Ariizumi, K., G. L. Shen, S. Shikano, S. Xu, R. Ritter, T. Kumamoto,

D. Edelbaum, A. Morita, P. R. Bergstresser, and A. Takashima (2000).

Identification of a novel, dendritic cell-associated molecule, dectin-1,

by subtractive cDNA cloning. J. Biol. Chem. 275, 20157�67.

26. Irjala, H., E. L. Johansson, R. Grenman, K. Alanen, M. Salmi, and

S. Jalkanen (2001). Mannose receptor is a novel ligand for L-selectin and

mediates lymphocyte binding to lymphatic endothelium. J. Exp. Med.

194, 1033�42.

27. Stahl, P. D. (1992). The mannose receptor and other macrophage lectins.

Curr. Opin. Immunol. 4, 49�52.

28. Hawiger, D., K. Inaba, Y. Dorsett, M. Guo, K. Mahnke, M. Rivera,

J. V. Ravetch, R. M. Steinman, and M. C. Nussenzweig (2001). Dendritic

cells induce peripheral T cell unresponsiveness under steady state

conditions in vivo. J. Exp. Med. 194, 769�79.

29. Chieppa, M., G. Bianchi, A. Doni, A. Del Prete, M. Sironi, G. Laskarin,

P. Monti, L. Piemonti, A. Biondi, A. Mantovani, M. Introna, and P. Allavena

(2003). Cross-linking of the mannose receptor on monocyte-derived

�187

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

dendritic cells activates an anti-inflammatory immunosuppressive

program. J. Immunol. 171, 4552�60.

30. Kwon, D. S., G. Gregorio, N. Bitton, W. A. Hendrickson, and D. R. Littman

(2002). DC-SIGN-mediated internalization of HIV is required for

trans-enhancement of T cell infection. Immunity 16, 135�44.

31. Ludwig, I. S., A. N. Lekkerkerker, E. Depla, F. Bosman, R. J. Musters,

S. Depraetere, Y. van Kooyk, and T. B. Geijtenbeek (2004). Hepatitis C

virus targets DC-SIGN and L-SIGN to escape lysosomal degradation.

J. Virol. 78, 8322�32.

32. Geijtenbeek, T. B. H., D. S. Kwon, R. Torensma, S. J. van Vliet,

G. C. F. van Duijnhoven, J. Middel, I. L. Cornelissen, H. S. Nottet,

V. N. KewalRamani, D. R. Littman, C. G. Figdor, and Y. van Kooyk

(2000). DC-SIGN, a dendritic cell-specific HIV-1-binding protein that

enhances trans-infection of T cells. Cell 100, 587�97.

33. Lozach, P. Y., A. Amara, B. Bartosch, J. L. Virelizier, F. Arenzana-Seisdedos,

F. L. Cosset, and R. Altmeyer (2004). C-type lectins L-SIGN and

DC-SIGN capture and transmit infectious hepatitis C virus pseudotype

particles. J. Biol. Chem. 279, 32035�45.

34. Alvarez, C. P., F. Lasala, J. Carrillo, O. Muniz, A. L. Corbi, and R. Delgado

(2002). C-type lectins DC-SIGN and L-SIGN mediate cellular entry by

Ebola virus in cis and in trans. J. Virol. 76, 6841�4.

35. Halary, F., A. Amara, H. Lortat-Jacob, M. Messerle, T. Delaunay, C. Houles,

F. Fieschi, F. Arenzana-Seisdedos, J. F. Moreau, and J. Dechanet-Merville

(2002). Human cytomegalovirus binding to DC-SIGN is required for

dendritic cell infection and target cell trans-infection. Immunity 17,

653�64.

36. Tassaneetrithep, B., T. H. Burgess, A. Granelli-Piperno, C. Trumpfheller,

J. Finke, W. Sun, M. A. Eller, K. Pattanapanyasat, S. Sarasombath,

D. L. Birx, R. M. Steinman, S. Schlesinger, and M. A. Marovich (2003).

DC-SIGN (CD209) mediates dengue virus infection of human dendritic

cells. J. Exp. Med. 197, 823�9.

37. Appelmelk, B. J., I. van Die, S. J. van Vliet, C. M. Vandenbroucke-Grauls,

T. B. Geijtenbeek, and Y. van Kooyk (2003). Cutting edge: carbohydrate

profiling identifies new pathogens that interact with dendritic cell-specific

ICAM-3-grabbing nonintegrin on dendritic cells. J. Immunol. 170, 1635�9.

38. Van Die, I., S. J. van Vliet, A. Kwame Nyame, R. D. Cummings, C. M. Bank,

B. Appelmelk, T. B. Geijtenbeek, and Y. van Kooyk (2003). The dendritic

cell specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni

egg antigens and recognizes the glycan antigen Lewis-x. Glycobiology

13, 471�8.

�

188

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

39. Cambi, A., K. Gijzen, J. M. de Vries, R. Torensma, B. Joosten, G. J. Adema,

M. G. Netea, B. J. Kullberg, L. Romani, and C. G. Figdor (2003). The C-type

lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida

albicans on dendritic cells. Eur. J. Immunol. 33, 532�8.

40. Geijtenbeek, T. B., S. J. van Vliet, E. A. Koppel, M. Sanchez-Hernandez,

C. M. Vandenbroucke-Grauls, B. Appelmelk, and Y. van Kooyk (2003).

Mycobacteria target DC-SIGN to suppress dendritic cell function. J. Exp.

Med. 197, 7�17.

41. Tailleux, L., O. Schwartz, J. L. Herrmann, E. Pivert, M. Jackson, A. Amara,

L. Legres, D. Dreher, L. P. Nicod, J. C. Gluckman, P. H. Lagrange,

B. Gicquel, and O. Neyrolles (2003). DC-SIGN is the major Mycobacterium

tuberculosis receptor on human dendritic cells. J. Exp. Med. 197, 121�7.

42. Baldari, C. T., A. Lanzavecchia, and J. L. Telford (2005). Immune

subversion by Helicobacter pylori. Trends Immunol. 26, 199�207.

43. Bergman, M. P., A. Engering, H. H. Smits, S. J. van Vliet,

A. A. van Bodegraven, H. P. Wirth, M. L. Kapsenberg,

C. M. Vandenbroucke-Grauls, Y. van Kooyk, and B. J. Appelmelk (2004).

Helicobacter pylori modulates the T helper cell 1/T helper cell 2 balance

through phase-variable interaction between lipopolysaccharide and

DC-SIGN. J. Exp. Med. 200, 979�90.

44. Appelmelk, B. J., M. A. Monteiro, S. L. Martin, A. P. Moran, and

C. M. Vandenbroucke-Grauls (2000). Why Helicobacter pylori has Lewis

antigens. Trends Microbiol. 8, 565�70.

45. Smits, H. H., A. Engering, D. van der Kleij, E. C. de Jong, K. Schipper,

T. van Capel, B. Zaat, M. Yazdanbakhsh, E. A. Wierenga, Y. Kooyk, and

M. L. Kapsenberg (2005). Selective probiotic bacteria induce regulatory

T cells by modulating dendritic cell function via DC-SIGN in vitro.

J. Allergy and Clin. Immunol. 115, 1260�7.

46. Geijtenbeek, T. B., P. C. Groot, M. A. Nolte, S. J. van Vliet,

S. T. Gangaram-Panday, G. C. van Duijnhoven, G. Kraal, A. J. van

Oosterhout, and Y. van Kooyk (2002). Marginal zone macrophages express

a murine homologue of DC-SIGN that captures blood-borne antigens

in vivo. Blood 100, 2908�16.

47. Baribaud, F., S. Pohlmann, and R. W. Doms (2001). The role of DC-SIGN

and DC-SIGNR in HIV and SIV attachment, infection, and transmission.

Virology 286, 1�6.

48. Taylor, P. R., G. D. Brown, J. Herre, D. L. Williams, J. A. Willment, and

S. Gordon (2004). The role of SIGNR1 and the beta-glucan receptor

(dectin-1) in the nonopsonic recognition of yeast by specific macrophages.

J. Immunol. 172, 1157�62.

�189

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

49. Kang, Y. S., J. Y. Kim, S. A. Bruening, M. Pack, A. Charalambous,

A. Pritsker, T. M. Moran, J. M. Loeffler, R. M. Steinman, and

C. G. Park (2004). The C-type lectin SIGN-R1 mediates uptake

of the capsular polysaccharide of Streptococcus pneumoniae

in the marginal zone of mouse spleen. Proc. Natl Acad. Sci. USA

101, 215�20.

50. Takahara, K., Y. Yashima, Y. Omatsu, H. Yoshida, Y. Kimura, Y. S. Kang,

R. M. Steinman, C. G. Park, and K. Inaba (2004). Functional comparison

of the mouse DC-SIGN, SIGNR1, SIGNR3 and Langerin, C-type lectins.

Int. Immunol. 16, 819�29.

51. Lanoue, A., M. R. Clatworthy, P. Smith, S. Green, M. J. Townsend,

H. E. Jolin, K. G. Smith, P. G. Fallon, and A. N. McKenzie (2004).

SIGN-R1 contributes to protection against lethal pneumococcal

infection in mice. J. Exp. Med. 200, 1383�93.

52. Nagaoka, K., K. Takahara, K. Tanaka, H. Yoshida, R. M. Steinman,

S. I. Saitoh, S. Akashi-Takamura, K. Miyake, Y. S. Kang, C. G. Park, and

K. Inaba (2005). Association of SIGNR1 with TLR4-MD-2 enhances

signal transduction by recognition of LPS in Gram-negative bacteria.

Int. Immunol. 17, 827�36.

53. Brown, G. D. and S. Gordon (2001). Immune recognition. A new

receptor for beta-glucans. Nature 413, 36�7.

54. Underhill, D. M., A. Ozinsky, A. M. Hajjar, A. Stevens, C. B. Wilson,

M. Bassetti, and A. Aderem (1999). The Toll-like receptor 2 is recruited

to macrophage phagosomes and discriminates between pathogens.

Nature 401, 811�15.

55. Brown, G. D., J. Herre, D. L. Williams, J. A. Willment, A. S. Marshall, and

S. Gordon (2003). Dectin-1 mediates the biological effects of beta-glucans.

J. Exp. Med. 197, 1119�24.

56. Gantner, B. N., R. M. Simmons, S. J. Canavera, S. Akira, and

D. M. Underhill (2003). Collaborative induction of inflammatory

responses by dectin-1 and Toll-like receptor 2. J. Exp. Med. 197, 1107�17.

57. Herre, J., A. S. Marshall, E. Caron, A. D. Edwards, D. L. Williams,

E. Schweighoffer, V. Tybulewicz, Reis E Sousa, S. Gordon, and

G. D. Brown (2004). Dectin-1 uses novel mechanisms for yeast

phagocytosis in macrophages. Blood 104, 4038�45.

58. Rogers, N. C., E. C. Slack, A. D. Edwards, M. A. Nolte, O. Schulz,

E. Schweighoffer, D. L. Williams, S. Gordon, V. L. Tybulewicz,

G. D. Brown, and Reis E Sousa (2005). Syk-dependent cytokine

induction by dectin-1 reveals a novel pattern recognition pathway

for C type lectins. Immunity 22, 507�17.

�

190

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

59. Gantner, B. N., R. M. Simmons, and D. M. Underhill (2005). Dectin-1

mediates macrophage recognition of Candida albicans yeast but not

filaments. EMBO J. 24, 1277�86.

60. d’Ostiani, C. F., G. Del Sero, A. Bacci, C. Montagnoli, A. Spreca,

A. Mencacci, P. Ricciardi-Castagnoli, and L. Romani (2000). Dendritic

cells discriminate between yeasts and hyphae of the fungus Candida

albicans. Implications for initiation of T helper cell immunity in vitro

and in vivo. J. Exp. Med. 191, 1661�74.

61. Romani, L., C. Montagnoli, S. Bozza, K. Perruccio, A. Spreca, P. Allavena,

S. Verbeek, R. A. Calderone, F. Bistoni, and P. Puccetti (2004).

The exploitation of distinct recognition receptors in dendritic cells

determines the full range of host immune relationships with Candida

albicans. Int. Immunol. 16, 149�61.

62. Huang, Q., D. Liu, P. Majewski, L. C. Schulte, J. M. Korn, R. A. Young,

E. S. Lander, and N. Hacohen (2001). The plasticity of dendritic cell

responses to pathogens and their components. Science 294, 870�5.

63. Jeannin, P., B. Bottazzi, M. Sironi, A. Doni, M. Rusnati, M. Presta,

V. Maina, G. Magistrelli, J. F. Haeuw, G. Hoeffel, N. Thieblemont,

N. Corvaia, C. Garlanda, Y. Delneste, and A. Mantovani (2005).

Complexity and complementarity of outer membrane protein A

recognition by cellular and humoral innate immunity receptors.

Immunity 22, 551�60.

64. Pinhal-Enfield, G., M. Ramanathan, G. Hasko, S. N. Vogel, A. L. Salzman,

G. J. Boons, and S. J. Leibovich (2003). An angiogenic switch in

macrophages involving synergy between Toll-like receptors 2, 4, 7,

and 9 and adenosine A(2A) receptors. Am. J. Pathol. 163, 711�21.

65. Mantovani A., S. Sozzani, M. Locati, P. Allavena, and A. Sica (2002).

Macrophage polarization: tumor-associated macrophages as a paradigm

for polarized M2 mononuclear phagocytes. Trends Immunol. 23, 549�55.

66. Mosser, D. M. (2003). The many faces of macrophage activation. J. Leukoc.

Biol. 73, 209�12.

67. Raes, G., L. Brys, B. K. Dahal, J. Brandt, J. Grooten, F. Brombacher,

G. Vanham, W. Noel, P. Bogaert, T. Boonefaes, A. Kindt, B. R. Van den,

P. J. Leenen, P. De Baetselier, and G. H. Ghassabeh (2005). Macrophage

galactose-type C-type lectins as novel markers for alternatively

activated macrophages elicited by parasitic infections and allergic

airway inflammation. J. Leukoc. Biol. 77, 321�7.

68. Higashi, N., K. Fujioka, K. Denda-Nagai, S. Hashimoto, S. Nagai, T. Sato,

Y. Fujita, A. Morikawa, M. Tsuiji, M. Miyata-Takeuchi, Y. Sano, N. Suzuki,

K. Yamamoto, K. Matsushima, and T. Irimura (2002). The macrophage

�191

PA

TH

OG

EN

-RE

CO

GN

ITIO

NR

EC

EP

TO

RS

AS

TA

RG

ET

SF

OR

PA

TH

OG

EN

ST

OM

OD

UL

AT

EIM

MU

NE

FU

NC

TIO

N

C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic

receptor expressed on monocyte-derived immature dendritic cells. J. Biol.

Chem. 277, 20686�93.

69. van Vliet, S. J., E. van Liempt, E. Saeland, C. A. Aarnoudse, B. Appelmelk,

T. Irimura, T. B. Geijtenbeek, O. Blixt, R. Alvarez, I. van Die, and Y. van

Kooyk (2005). Carbohydrate profiling reveals a distinctive role for the

C-type lectin MGL in the recognition of helminth parasites and tumor

antigens by dendritic cells. Int. Immunol. 17, 661�9.

�

192

A.

EN

GE

RIN

G,

S.J

.V

AN

VL

IET,

E.A

.K

OP

PE

LET

AL.

CHAPTER 10

Suppression of immune responses by bacteriaand their products through dendritic cellmodulation and regulatory T cell induction

Miriam T. Brady, Peter McGuirk and Kingston H. G. MillsTrinity College, Dublin

10.1 INTRODUCTION

Infection with pathogenic bacteria can result in acute or chronic disease,

which can be life threatening, especially in young, elderly or other

immunocompromised individuals. Humans are also infected with a wide

range of commensal bacteria, as part of our normal gut flora, and the

immune system must be capable of controlling immune responses against

these beneficial bacteria, while at the same time generating effector

immune responses against pathogenic micro-organisms. In addition,

pathogenic bacteria have evolved strategies for delaying or preventing their

elimination by evading or subverting protective immune responses of

the host.

10.1.1 Innate immunity to bacteria

The initial inflammatory response to pathogenic bacteria involves the

release of cytokines and chemokines and the recruitment of neutrophils,

monocytes, dendritic cells (DCs) and lymphocytes to the site of infection.

Tissue macrophages and neutrophils quickly phagocytose and attempt

to kill the bacteria. Macrophages and DCs are activated through binding

of conserved, secreted or cell surface bacterial products to pathogen

recognition receptors (PRR). This leads to activation of immune response

genes, including those coding for inflammatory cytokines, chemokines

and co-stimulatory molecules expressed on the surface of DCs

and macrophages, that are involved in antigen presentation (Janeway and

Medzhitov, 2002).

Bacteria are phagocytosed by neutrophils and macrophages and this

is facilitated through activation of the alternative complement pathway

by bacterial cell wall components, resulting in the production of C3b,

which together with antibodies help to opsonize the bacteria. Once inside

the phagocytic cells, the bacteria-containing phagosome undergoes a series

of fusions with endosomes and lysosomes to form a phagolysosome where

the bacteria are killed in a variety of ways, including reactive oxygen

and nitrogen-dependent mechanisms. However, bacteria can inhibit kill-

ing by preventing phagosome�lysosome fusion or by inducing lysosomal

discharge into the cytoplasm. Induction of apoptosis in neutrophils,

macrophages and DCs, critical cells in anti-bacterial host defence and in

activating adaptive immunity, is an alternative immune evasion strategy

evolved by a number of bacterial species to subvert protective immune

responses of the host.

10.1.2 Adaptive immunity to bacteria

Following uptake of the bacteria by a macrophage or DC, these pro-

fessional antigen presenting cells (APC) process and present peptides of

the bacterial antigens to T cells, in association with major histocom-

patibility complex (MHC) molecules on their cell surface. All bacteria,

whether they survive intracellularly or extracellularly in the host, can

activate MHC class II restricted CD4þ T helper (Th) cells. In addition,

certain intracellular bacteria can also activate MHC class I-restricted CD8þ

cytotoxic T lymphocytes (CTL). Induction of class II-restricted T cells

requires processing by an exogenous route. Class I restricted CTL kill

host cells infected with intracellular bacteria, whereas the main function

of class II-restricted CD4þ T cells is to release cytokines that activate

phagocytosis and killing of bacteria by macrophages and to provide helper

function for antibody production. CD4þ T cells can be divided into

a number of functionally distinct subtypes discriminated on the basis of

cytokine secretion. Th1 cells secrete interferon-g (IFN-g) and tumor

necrosis factor-b (TNF-b) and activate phagocytosis and killing by macro-

phages and provide help for the production of opsonizing (murine IgG2a)

antibodies. More recently a distinct population of effector T cells that

secrete IL-17 and promote inflammatory reactions in autoimmune diseases

also appear to play a protective role in immunity to certain bacteria (Happel

et al., 2003). Th2 cells secrete IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13

and are considered to be the main helper cells, especially for IgG1, IgA

and IgE. Finally, T cells that secrete IL-10 and/or TGF-b, termed regulatory

�

194

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

T (Treg) cells, that suppress immune responses mediated by innate

immune cells and other T cells, can be induced by infection (Mills, 2004).

The control of intracellular bacteria is dependent on the induction

of cell-mediated immunity, however, humoral immunity plays a major

role in protection against extracellular bacteria. In individuals that have

recovered from self-limiting bacterial infections, antibodies play a major

role in preventing re-infection. Furthermore successful bacterial vaccines

confer protection by the generation of circulating IgG or memory B cells,

which produce an anamnestic antibody response following re-exposure

to the bacteria. However, the generation of antibody responses is also

dependent on priming of helper T cells, so both T and B cells are critical

in the primary as well as secondary response to bacterial infection. Ig class

switching to IgG2a in the mouse is promoted by IFN-g-secreting

Th1 cells and this antibody subclass is involved in opsonization of bacteria

and also stimulates complement components C3b and iC3b, which bind

complement receptors and further promote phagocytosis. Extracellular

bacteria often bind to epithelial cells in the respiratory or gastrointestinal

(GI) tract via adhesins or pili, and antibodies against these bacterial virulence

factors can help to prevent colonization. Antibodies of the murine IgG1

subclass can neutralize bacterial toxins and prevent their binding to host

target cells and thereby reduce the severity of diseases caused by toxin-

producing bacteria. IgA antibodies induced following bacterial infection

of the respiratory tract, GI tract or other mucosal tissues, function to limit

the infection to mucosal surfaces.

In addition to CD4þ and CD8þ Tcells, unconventional T cells, including

gd T cells which recognize phospholigands and CD1-restricted ab T cells,

or T cells that express natural killer (NK) markers, termed NKT cells,

that recognize glycolipids, also play a role in immunity to intracellular

bacteria (Schaible and Kaufmann, 2000). Although it takes days rather

than hours before the adaptive immune response is effective, unlike the

innate immune system, it is able to recall previous encounters with antigen,

through the activation of memory T and B cells. Therefore a second or

subsequent infection with the bacteria is dealt with more effectively.

10.2 T CELL ANERGY OR SUPPRESSION INDUCED

BY BACTERIA AND THEIR PRODUCTS

T cells play a major role in clearance of a primary infection with patho-

genic bacteria and are fundamental in the memory response induced

by previous infection or vaccination. However in many bacterial infections,

�195

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

T cell responses either fail to develop, are suppressed, anergic or are

skewed to an inappropriate subtype required for bacterial elimination. T cell

activation is dependent on three signals. Signal one is provided through

engagement of the T cell receptor (TCR) with processed antigenic peptide

associated with a MHC molecule expressed on the surface of an APC.

Signal two is provided by interaction of CD28 on the T cell surface with

co-stimulatory molecules, CD80 or CD86 expressed on the surface of

activated APC. In the absence of the second signal the T cells become

anergic and are no longer able to divide or respond to antigen. A third

signal which determines the polarization of the T cell response to distinct

subtypes is provided by regulatory cytokines, such as IL-4, IL-6, IL-10, IL-12,

IL-23 and IL-27, secreted primarily by cells of the innate immune system.

Many bacteria have evolved strategies to evade adaptive immunity by

suppressing various steps in T cell activation or polarization, or by inducing

a state of anergy, whereby T cells are unable to respond to the foreign

pathogen. Interference with any aspect of antigen uptake processing

or presentation by the APC, or signaling in the T cell can inhibit T cell

activation. Furthermore, since activation of distinct T cell subtypes are

regulated by the reciprocal subtype, factors that strongly promote the

induction of one subtype may inhibit the activation of another. The Th1/

Th2 paradigm has provided a simple model to explain the persistence of

certain bacterial infections through pathogen induction of the reciprocal

subtype; IFN-g secreted by Th1 cells can suppress Th2 responses, whereas

IL-4 and IL-10 secreted by Th2 cells can suppress Th1 responses. However,

recent advances in our understanding of the role of Treg cells has

complicated this model and it now appears that natural and inducible

Treg cells may play a critical role in controlling both Th1 and Th2 cells and

may also be responsible for anergy or immunosuppression observed

during certain infections (Mills, 2004).

Salmonella typhimurium infection of mice results in profound

immunosuppression, with inhibition of T and B cell proliferation and IL-2

production in response to foreign antigen and mitogens (al-Ramadi et al.,

1991a,b). Suppression with S. typhimurium was linked with soluble

factors released by monocytes/macrophages and was reversed by the

addition of IL-4. Nitric oxide (NO) was later shown to be involved in the

immunosuppression as treatment of mice with the NO inhibitor, amino-

guanidine hemisulfate, blocks the suppressive effect of Salmonella infec-

tion on T and B cell responses (MacFarlane et al., 1999). However the NO

inhibitor also blocked the influx of neutrophils and macrophages into

the spleen and enhanced bacterial load, resulting in higher mortality

�

196

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

of the mice, suggesting that NO is involved in host defence.

Helicobacter pylori infection is also associated with immunosuppression

and in vitro studies demonstrated that H. pylori suppressed proliferative

responses of human PBMC to mitogens and antigens. A soluble cytoplasmic

fraction of H. pylori was found to mediate the suppression by acting on

monocytes and directly on the T cells (Knipp et al., 1994).

In certain bacterial infections where immunosuppression has been

reported, the bacterial virulence factor involved has been identified. The

YopH protein of Yersinia pseudotuberculosis, which has tyrosine phos-

phatase activity, has been shown to suppress immune responses by

interfering with T and B cell antigen-receptor activation (Yao et al., 1999).

The OspA protein of Borrelia burgdorferi inhibits proliferative responses of

human PBMC to mitogens (Chiao et al., 2000). Furthermore B. burgdorferi

infection of disease susceptible (C3H/HeJ) and resistant (BALB/c) mice

results in impaired proliferation, and IL-2 and IL-4 production to mitogens

(de Souza et al., 1993). More recently it has been suggested that Th1

responses are suppressed while Th2 responses are enhanced; B. burgdorferi

transmission by Ixodes scapularis suppressed IL-2 and IFN-g and enhanced

IL-4 production in mice (Zeidner et al., 1997). Similarly, the inhibitory

effect of Escherichia coli LT on T cell responses was shown to be specific

for Th1 cells and was mediated by the effect of ADP-ribosyl transferase

enzyme activity on APC as well as on T cells (Ryan et al., 2000). However,

recent studies with the related AB type toxin, cholera toxin (CT), have

suggested that that these toxins induce suppressive Treg cells, as well as

Th2 cells (Lavelle et al., 2003). It has also been reported that E. coli

heat labile enterotoxin (LT)-treated epithelial cells release soluble factors,

probably prostaglandins, that inhibit T cell proliferation in vitro (Lopes

et al., 2000).

Patients with lepromatous leprosy are highly immunosuppressed;

their T cells do not respond to Mycobacterium leprae antigens and are

anergized to unrelated antigens (Mehra et al., 1984; Salgame et al., 1984). The

immunosuppression has been linked to macrophages, CD8þ T cells,

inappropriate Th1/Th2 induction or IL-10 production. Lipoarabinomannan

(LAM)-B protein from M. leprae can suppress proliferative responses of

PBMC from lepromatous leprosy patients, tuberculoid leprosy patients

and from normal individuals (Kaplan et al., 1987). Lipoglycans from M. leprae

have also been shown to induce immune suppression in mice, including

inhibition of delayed type hypersensitivity responses and proliferation of

lymph node cells to mitogens (Moura et al., 1997). In contrast to patients

with lepromatous leprosy, where T cell responses to many antigens are

�197

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

profoundly suppressed and the bacteria persist, patients with tuberculoid

leprosy have potent cellular immune responses and control the infection

(Modlin, 1994). The patients with lepromatous leprosy mount Th2

responses, which are not protective, whereas in the tuberculoid leprosy

patients Th1 cells are dominant and IFN-g secreted by these cells activate

infected macrophages to kill the bacteria.

Inappropriate Th2 cell induction and/or suppression of Th1 cells

have also been shown to result in increased susceptibility to bacterial

infections. Progression of B. burgdorferi infection following transmission

by I. scapularis, in disease-susceptible C3H/HeJ mice is associated with

the development of Th2 responses, but not in disease-resistant BALB/c

mice (Zeidner et al., 1997). IFN-g secreted by NK cells during the acute

stages of infection, and by Th1 cells later in infection, have been shown to

play a crucial role in the clearance of Bordetella pertussis from the respiratory

tract; mice depleted of NK cells or with defective IFN-g receptors develop

disseminating lethal infections (Byrne et al., 2004; Mahon et al., 1997).

However, B. pertussis infection of immunocompetent individuals persists

for several weeks or months. Furthermore, studies in a mouse model

have shown that Th1 responses are suppressed in the lungs and draining

lymph nodes during acute infection and this has been linked to the

induction of innate anti-inflammatory cytokines and Treg cells by B. pertussis

virulence factors (McGuirk et al., 2002; McGuirk and Mills, 2000; Ross

et al., 2004).

10.3 MODULATION OF DC CYTOKINE PRODUCTION

BY BACTERIA AND BACTERIAL MOLECULES

Dendritic cells utilize TLRs and other PRRs, including C-type lectins, to

recognize characteristic conserved molecular patterns in microbial cell-wall

components. Binding of PRRs on DCs with these pathogen associated

molecular patterns (PAMPs) and consequent antigen capture, results in

a cascade of events ultimately resulting in the activation of an appropriate

immune response and elimination of the pathogen.

Following antigen capture in peripheral mucosal tissues, DCs migrate

to secondary lymphoid organs for efficient antigen presentation. Concomi-

tantly DCs undergo considerable changes including the activation of

immune response genes, inflammatory cytokine and chemokine production,

and provision of appropriate co-receptor signaling required for effective

T-cell stimulation (Janeway and Medzhitov, 2002). Different microbial

stimuli confer distinctive signals to the DCs via PRRs, modulating the

�

198

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

DC response and the subsequent differentiation of naive T-cells. In

addition, pro-inflammatory cytokines and chemokines such as TNF-a,

IL-1, IL-12 and MIPs promote the infiltration of leucocytes to the site of

infection following bacterial challenge. These cytokines also play a crucial

role in enhancing the bactericidal activity of phagocytes and in directing

the immune response through the modulation of DCs. In this way the

induction of protective immunity by DCs is highly dependent on the

stimulus received and cytokines induced, and is therefore susceptible to

manipulation by pathogens, which have evolved mechanisms to subvert

DC function and escape immune surveillance. This immune regulation

is a universal concept that includes suppression, diversion and conversion

of the immune response to the benefit of the pathogen.

10.3.1 Suppression of IL-12 and TNF-a

A common strategy utilized by bacteria to modulate host responses

is to suppress the immediate inflammatory response, normally associated

with the production of proinflammatory cytokines, such as IL-12 and

TNF-a. This is achieved either through the production of IL-10, TGF-b,

or interference with signaling pathways.

Immunosuppression is an inherent complication of mycobacterial

infections. Several studies have shown that M. tuberculosis or M. bovis

bacillus Calmette-Guerin (BCG) target the C-type lectin DC-SIGN

(DC-specific intercellular adhesion molecule-grabbing nonintegrin) to

infect DCs and inhibit their immunostimulatory function (Geijtenbeek

et al., 2003; van Kooyk and Geijtenbeek, 2003). This is thought to

occur through the interaction of the mycobacterial mannosylated LAM

with DC-SIGN, which can inhibit IL-12 production (Gagliardi et al., 2005;

Nigou et al., 2001), and prevent DC maturation (Geijtenbeek et al., 2003)

(see also Chapter 9). However, recently it has been shown that BCG-

induced impairment of IL-12 from DCs cannot be attributed to the sole

engagement of this receptor, as it occurs irrespective of DC-SIGN

expression (Gagliardi et al., 2005).

Filamentous hemagglutinin (FHA) is an adhesin molecule and

virulence factor from B. pertussis, which binds to the b2 integrin CR3

(Ishibashi et al., 1994). This bacterial molecule which interacts directly with

DCs and macrophages also has immunomodulatory properties and has

been shown to inhibit IL-12 production, partly through induction of IL-10

(McGuirk et al., 2002; McGuirk and Mills, 2000). In addition, FHA is capable

of inhibiting both the Th1 response to influenza virus when administered

�199

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

simultaneously in the respiratory tract (McGuirk et al., 2000), and LPS-driven

IL-12 and IFN-g production in vivo, in a murine model of septic shock

(McGuirk and Mills, 2000). The ability of FHA to suppress IL-12 production

is particularly relevant given that during acute infection, antigen-specific Th1

responses in the lung and its draining lymph nodes are also severely

suppressed (McGuirk and Mills, 2000).

A number of bacterial toxins can also inhibit inflammatory responses

by cells of the innate immune system. They include the AB-type toxins CT

from Vibrio cholerae (Braun et al., 1999), adenylate cyclase toxin (CyaA)

from B. pertussis (Mills, 2001) and E. coli LT (Ryan et al., 2000). CT has

the ability to prevent the production of bioactive IL-12 and expression of

the IL-12 receptor b1 and b2 chains on human monocytes and DCs (Braun

et al., 1999), and can inhibit IL-12-mediated experimental colitis (Boirivant

et al., 2001). Similarly, CT inhibits LPS or CD40L-induced IL-12 and TNF-aand chemokine production by human DC (Gagliardi et al., 2000). LT, and

the partially toxic mutant LTR72 suppress production of IL-12 and TNF-aand the inflammatory chemokines MIP-1a and MIP-1b (Ryan et al., 2000).

Another LT mutant, LTK63, which is devoid of ADP-ribosylating activity,

does not possess all the inhibitory properties of the wild-type or partially

toxic mutant, suggesting that enzyme activity is in part responsible for

suppression. The proinflammatory cytokine inhibition by both CT and LT

has subsequently been shown to be mediated through a cAMP-dependent

mechanism (Bagley et al., 2002a; Ryan et al., 2000). CyaA from B. pertussis

can also mediate anti-inflammatory effects through cAMP, by inhibiting

IL-12 and TNF-a production by human monocyte-derived DCs (Bagley

et al., 2002b), and has also been shown to inhibit LPS-induced IL-12 and

TNF-a production from murine DCs (Ross et al., 2004).

The major virulence factors from Bacillus anthracis, the causative

agent of anthrax, are the toxins lethal toxin and edema toxin and are

both inhibitory. Prestimulation of murine DCs with purified lethal toxin

severely impairs IL-12, TNF-a, IL-1b and IL-6 production after sub-

sequent stimulation with LPS, and impairs their ability to prime allo-

geneic CD4þ T-cells (Agrawal et al., 2003). Edema toxin, another ADP-

ribosylating toxin, which exerts anti-inflammatory effects by increasing

intracellular cAMP levels (Hoover et al., 1994), has recently been shown

to cooperate with lethal toxin to impair cytokine secretion during

infection of DCs (Tournier et al., 2005). Murine bone marrow-derived

DC exhibit very different cytokine secretion patterns when infected

with B. anthracis strains secreting lethal toxin or edema toxin or both,

and when infected with a lethal toxin/edema toxin negative strain.

�

200

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

Edema toxin inhibits IL-12p70 and TNF-a secretion, whereas lethal toxin

inhibits IL-10 and TNF-a production. Simultaneous secretion of both

lethal toxin and edema toxin have a cumulative effect on the inhibition

of TNF-a (Tournier et al., 2005). Lethal toxin and edema toxin

display dominant effects on IL-12p70 and IL-10, respectively, and

therefore during infection, co-function to impair DC cytokine secretion.

Pasteurella multocida toxin (PMT), which is a major virulence factor

in progressive atopic rhinitis in wild and domestic animals has also

recently been demonstrated to suppress IL-12 production in human

monocyte-derived DCs, and inhibit the mucosal adjuvant effects of CT

in mice (Bagley et al., 2005). TNF-a production is suppressed at the site

of infection and in the Peyer’s patches of mice infected with Yersinia

enterocolitica (Beuscher et al., 1995). Infection of DCs with wild-type

Y. enterocolitica also suppresses the release of TNF-a, IL-10 and IL-12,

while infection with plasmid-cured Y. enterocolitica or with a YopP-deficient

mutant resulted in the production of these cytokines (Erfurth et al., 2004).

Probiotics, which are defined as microbial organisms beneficial to the

host following ingestion, are now widely accepted as having immuno-

modulatory properties and playing an important role in the maintenance

of homeostasis. In a recent study, it has been demonstrated that various

Lactobacilli species can exert differing and opposing effects on DC

activation (Christensen et al., 2002). DCs treated with L. reuteri secrete

high levels of IL-6 and IL-10 in contrast to L. casei-treated DCs which

preferentially induce IL-12 production (Christensen et al., 2002). The anti-

inflammatory effect of some Lactobacilli has been most efficiently demon-

strated in the treatment of colitis. A higher proportion of Lactobacillus

species that inhibit TNF-a can be recovered from mice without colitis

compared to mice with microbiota-dependent colitis (IL-10-deficient) (Pena

et al., 2004). Furthermore, treatment of H. hepaticus-induced colitis with

L. reuteri and L. paracasei, lowers proinflammatory colonic cytokine (IL-12

and TNF-a) levels, and reduces intestinal inflammation (Pena et al., 2005).

L. reuteri and L. rhamnosus have also shown to be beneficial in the

management of atopic dermatitis in children (Rosenfeldt et al., 2003).

10.3.2 Induction of IL-10 and TGF-b

The anti-inflammatory cytokine, IL-10, which has the ability to inhibit

responses of T-cells and macrophages and other cell types, plays a key

role in immune regulation by microbial pathogens. Its principal function

appears to be to limit and ultimately terminate inflammatory responses

�201

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

(Moore et al., 2001), but has been shown to be exploited by pathogens to

delay their elimination from the host. These effects are primarily mediated

via the modulation of APC function (McBride et al., 2002; Redpath et al.,

2001). A number of bacteria including B. pertussis (McGuirk et al., 2002),

Mycobacterium spp. (Redpath et al., 2001), Yersinsia spp. (Erfurth et al.,

2004), Listeria spp. (Brzoza et al., 2004) and Lactobacilli (Smits et al.,

2005), have been shown to induce IL-10 production from macrophages

or DCs.

The perturbation of cytokine networks is increasingly recognized as

a pathogenicity mechanism exploited by a number of bacteria. However,

the bacterial molecule(s) responsible has only been identified in a limited

number of cases. FHA, an adhesin from B. pertussis which binds CR3

(Ishibashi et al., 1994), induces IL-10 production from DC (McGuirk

et al., 2002; McGuirk and Mills, 2000). Y. enterocolitica rLcrV (virulence

associated V antigen) or infection with Y. enterocolitica can suppress

TNF-a production in mice (Beuscher et al., 1995; Sing et al., 2002). This

process has been attributed to IL-10 induction by the bacteria, since mice

lacking IL-10 have significantly increased survival following infection,

have lower bacterial numbers, and a distinctive absence of TNF-asuppression (Sing et al., 2002). Furthermore, it has been suggested that

induction of IL-10 from DCs after infection with Y. enterocolitica may

contribute to the reduction in T-cell proliferation observed in vitro (Erfurth

et al., 2004).

Immunosuppression associated with mycobacteria and the sup-

pressed type 1 responses of M. tuberculosis infected individuals has

been attributed to the production of IL-10 and TGF-b production by

mononuclear cells, including macrophages and DCs. These inhibitory

cytokines have been implicated in the hyporesponsiveness observed in

the lungs of patients with active pulmonary TB, providing a microenviron-

ment wherein immune cells become refractory to appropriate activating

signals (Bonecini-Almeida et al., 2004). LAM, a cell wall component of

M. tuberculosis, induces IL-10 production from human mononuclear cells

and DCs (Barnes et al., 1992; Chieppa et al., 2003), and TGF-b from

human monocytes (Dahl et al., 1996), and this has also been provided

as an explanation for the immunosuppressive effects of the bacteria on

antigen-induced T-cell proliferation. LAMs from M. tuberculosis have also

been shown to inhibit LPS-induced IL-12p70 and p40 production from

human monocyte-derived DCs, an effect which is dependent on the presence

of mannooligosaccharide caps (Nigou et al., 2001). LAMs may therefore act as

virulence factors for the bacteria, inducing anti-inflammatory cytokines and

�

202

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

preventing the induction of IL-12, which is critical for the generation of Th1

responses required to effectively eliminate M. tuberculosis. M. tuberculosis

secretory antigen can down-regulate Th1 responses to mycobacteria by

differentially modulating the cytokine profiles of DCs. Restoration of the

attenuated Th1 response can be achieved by blocking IL-10 or TGF-b with

monoclonal antibodies, indicating a pivotal role for these cytokines in

suppression of immune responses to mycobacteria (Balkhi et al., 2004;

Natarajan et al., 2003). Similarly, suppression of IFN-g production by purified

protein derivative (PPD) from M. tuberculosis is abrogated following

neutralization of IL-10 and TGF-b (Othieno et al., 1999).

Studies in IL-10 knockout (IL-10�/�) mice have provided more definitive

evidence of an important role for endogenous or bacteria-stimulated IL-10

production on the course of bacterial infections. In certain cases infection

is less severe in the absence of IL-10, confirming that this cytokine may

subvert protective immunity by suppressing innate and adaptive immune

responses. For example, lack of IL-10 during M. bovis BCG infection leads

to accelerated clearance of bacilli, and enhanced plasma concentrations

of the inflammatory cytokines IL-12 and TNF-a (Jacobs et al., 2000; Jacobs

et al., 2002). Furthermore, in the absence of IL-10, BCG-infected DCs

are more effective at trafficking to lymph nodes, produce significantly

more IL-12, and enhance IFN-g production in response to mycobacterial

antigens compared to wildtype DCs (Demangel et al., 2002). IL-10�/�

mice are highly resistant to Yersinia infection and lack the IL-10-mediated

TNF-a suppression observed in susceptible animals (Sing et al., 2002).

However, in other infections the role of IL-10 in terminating potentially

adverse effects of inflammatory responses has been highlighted, by the

observation that severity of disease is exacerbated in absence of this

cytokine. In cerebral infection with Listeria monocytogenes, IL-10�/� mice

succumb to primary and secondary infection, and recruit significantly

more inflammatory cells to the brain. Furthermore, inflammatory cytokine

production such as IL-1b, TNF-a and IL-12, and severity of brain

lesions is enhanced in IL-10�/� mice (Deckert et al., 2001). Despite this

prominent hyperinflammation, intracerebral bacterial load is not reduced

in IL-10�/� mice compared with wildtype mice, suggesting that IL-10 in

this case plays a more significant role in damage limitation rather than

protective immunity. However, TGF-b has been shown to protect against

lethal infection and inhibit the production of inflammatory cytokines during

listeriosis (Nakane et al., 1996).

There are also examples of bacterial infection in IL-10�/� mice where

disease pathology is enhanced, or where bacterial clearance is accelerated.

�203

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

Colonization of the gastric mucosa by Helicobacter pylori is significantly

reduced in IL-10�/� mice, but infiltration of inflammatory mediators,

and severity of chronic active gastritis is enhanced compared to WT

mice (Chen et al., 2001). Similarly, endogenous IL-10 impairs bacterial

clearance during E. coli peritonitis. However, despite lower bacterial

numbers, IL-10�/� mice had higher concentrations of inflammatory

cytokines and demonstrated more severe organ damage (Sewnath et al.,

2001). Hence, although IL-10 may enhance dissemination of the bacteria,

it protects mice from lethality by attenuating immune-mediated pathology.

IL-10�/� mice infected with Helicobacter hepaticus develop chronic colitis,

with a critical role for IL-12 in both the induction and maintenance

of the inflammatory process following infection (Kullberg et al., 2001).

In wild-type mice, infection with H. hepaticus induces Treg cells that

prevent bacteria-induced colitis (Kullberg et al., 2002). However, in the

absence of IL-10 this protective mechanism fails, and mice develop chronic

disease.

10.4 INFLUENCE OF BACTERIA AND BACTERIAL MOLECULES

ON DC MATURATION

Dendritic cells are pivotal in the activation of naive T-cells and for the

initiation of the primary immune response. The factors that determine DC

function depend on the nature of the pathogen, the maturation status

induced and the local environment. The process of maturation involves

the continuous transition from an immature DCs residing in peripheral

tissue and sampling its environment, to the mature DCs in the secondary

lymphatic organs. These mature DCs are fully equipped for the efficient

stimulation of Ag-specific T-cells.

Steady-state DCs are immature, have high phagocytic activity, express

low-levels of costimulatory molecules and have low antigen processing

capacity (Banchereau et al., 2000). Microbial-induced maturation signals,

such as LPS, TNF-a, CD40 ligand and antigen interactions instigate the

maturation process. Depending on the initial signals or stimuli provided

by the pathogen, various signaling pathways are triggered within the

DCs, which then undergo considerable modification, such as induction

of costimulatory molecules, cytokine production, migratory properties and

endocytic activity or morphology. Additional DC stimulation is achieved

and modulated by proinflammatory cytokines such as TNF, IL-1b and

prostaglandin E2, or by a variety of non-inflammatory and pathogen

unrelated factors like histamine and ATP. These supplementary stimuli

�

204

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

influence the nature of a maturational process and provide DCs with

different capacities for T cell effector subset priming (Kalinski et al.,

1999; Lanzavecchia and Sallusto, 2001). The upregulation of MHC and

costimulatory molecules enhances the antigen presenting capacities of

DCs and their T-cell stimulatory potential. This complex process culmi-

nates in the arrival of activated DCs in the T cell zone of lymph nodes

where they interact with T cells. Therefore, the bacterial antigens that

are able to induce maturation of DCs, and the extent of this maturation,

help to define the character of primary immune responses against the

pathogen and thus have an important role in determining the course

of an infection (Reis e Sousa, 2004).

In addition to the modulation of DC cytokine production (discussed

above), bacteria or their various virulence factors can also modulate MHC

and co-stimulatory molecule expression on DCs. The AB-type toxins CT

and LT induce maturation of human monocyte-derived DCs, with upregula-

tion of CD80, CD86, CD83 and HLA-DR (Bagley et al., 2002a). This

maturation process is at least in part dependent on elevation of intracellular

cAMP levels, which the toxins achieve through the constitutive activation of

adenylate cyclase. Enzymatically inactive toxins such as CTK63 and LTK63

are unable to upregulate cAMP levels in the cell, and thus are less efficient in

activating DCs. In addition, phenotypic maturity as determined by enhanced

surface marker expression, correlates with an increased ability to stimulate

proliferation of allogeneic T-cells. However, although the AB toxins mature

DCs with regard to phenotype and functionality, they also profoundly inhibit

IL-12 and TNF-a production (Bagley et al., 2002a). CT has been shown to

mature DCs which prime Th2 cells (Anjuere et al., 2004; Gagliardi et al.,

2000), which may be due to its capacity to suppress the Th1-driving cytokine

IL-12. CT also has the ability to promote the induction of Treg cells against

bystander antigens, by modulating DC activation (Lavelle et al., 2003).

Stimulation of DC with CT enhanced expression of CD80, CD86, but

downregulated CD40 expression, which is known to be important for

induction of IL-12 and Th1 responses. Furthermore, adoptive transfer of

DCs pulsed with antigen in the presence of CT, primed antigen-specific

T-cells that produced IL-10, low levels of IL-4 and IL-5, and almost

undetectable IFN-g, a Tr1 type cell phenotype.

Bacillus anthracis, the causative agent of anthrax, has the ability to

disrupt host immune responses by specifically targeting DCs. One of its

critical virulence factors is anthrax lethal toxin, which not only modu-

lates DC cytokine production but has a profound effect on the func-

tional capacity of DCs (Agrawal et al., 2003; Tournier et al., 2005).

�205

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

Upregulation of CD40, CD80 and CD86 on DCs is severely impaired

following exposure to lethal toxin and these DCs fail to efficiently prime

allogeneic CD4þ T-cells and are refractory to further stimulus by the

potent mitogen PMA and ionomycin (Agrawal et al., 2003). The mechanism

involved was shown to involve impaired phosphorylation of p38 and

ERK1/ERK2. DC function is also perturbed during infection with

Y. enterocolitica. Bacteria invade DC at the onset of infection and down-

regulate MHC class II and CD80 molecules, which correlates with a

decreased T-cell activation capacity (Schoppet et al., 2000). The decrease

in immunostimulatory ability is a transient effect, but may be sufficient

to facilitate bacterial persistence in susceptible individuals. Similar effects

have been observed in murine DCs (Erfurth et al., 2004).

Coxiella burnetii, the etiological agent of the zoonotic disease Q fever,

can evade immune clearance and result in a persistent infection. This

pathogen has been shown to evade the protective response through

manipulation of DC maturation. A virulent strain can infect human

DCs but does not result in their maturation, demonstrated by the lack

of inflammatory cytokine production, and upregulation of surface markers

CD80, CD86, CD40, CD83 and HLA-DR. In contrast, an avirulent

strain which possesses a severely truncated LPS, effectively induces DC

maturation (Shannon et al., 2005). The lack of DC maturation by the

virulent strain results in an immune response that impedes clearance of

the bacteria and allows persistence. The mechanism proposed is that the

intact LPS of the virulent strain ‘‘masks’’ TLR ligands from recognition

by DCs, preventing DC maturation and induction of the appropriate

immune response for bacterial clearance.

10.5 TREG CELLS IN BACTERIAL INFECTIONS

10.5.1 Natural and inducible regulatory T cells

A population of CD4þ T cells, that secrete high levels of IL-10 and/or

TGF-b, but low or undetectable IL-4 and IFN-g, termed inducible Treg

cells have recently been described during infection with certain patho-

gens, including bacteria (McGuirk et al., 2002). Treg cells have suppressive

function and are induced by the host to control immune responses during

infection, especially Th1 responses, and thereby limit infection-induced

immunopathology (Mills, 2004). Alternatively their induction may serve

as an evasion strategy by the bacteria to suppress protective Th1 responses.

�

206

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

Another population of suppressor T cells, initially characterized

through their expression of CD25, but more recently by the transcriptional

repressor Foxp3, termed natural Treg cells also play a crucial role in

regulating immune responses (Hori and Sakaguchi, 2004). These cells

which account for 5�50 per cent of circulating T cells were shown by

depletion and adoptive transfer experiments to be capable of preventing

autoimmune diseases through their ability to maintain tolerance to self

antigens (Sakaguchi, 2000). CD25þ T cells have been shown in a number

of systems to be capable of suppressing proliferation and cytokine secretion

by CD4þCD25� T cells. Natural Treg cells also play a role in mucosal

tolerance and help to control immune responses to inhaled antigens in

the lung (Ostroukhova et al., 2004) and to commensal bacteria in the

gut (Powrie, 2004). In addition there is evidence that they function to regulate

protective immunity to pathogens, including pathogenic bacteria (Mills,

2004).

10.5.2 Treg cells induced by bacteria modulated DC

It has been suggested that the activation status of the DC, rather than

the lineage, determines its ability to selectively promote T cell subtypes

(Barrat et al., 2002). Many TLR ligands, including LPS, CpG motifs

in bacterial DNA and viral dsRNA have been shown to promote IL-12

production, and to activate DC that promote the differentiation of Th1

cells (Hemmi et al., 2000; Moser and Murphy, 2000) (Figure 10.1).

In contrast, products of helminth parasites, yeast hyphae and CT activate

DCs, which directs the induction of Th2 cells (d’Ostiani et al., 2000; Gagliardi

et al., 2000; Moser and Murphy, 2000). Since inducible Treg cells (Tr1 and Th3

cells) arise from naive or resting CD4þ T cells in the periphery, it was highly

conceivable that DCs activated with an appropriate stimulus, such as certain

pathogen-derived molecules, could selectively promote the induction of Treg

cells. Indeed, evidence is emerging that bacteria or bacteria-derived

molecules can promote the induction of Tr1 cells, through their interaction

with DCs or other innate cells, such as macrophages.

The induction of high levels of IL-10 and/or TGF-b by DCs following

infection has been reported for a number of bacterial species. In certain

cases, specific pathogen-derived immunoregulatory molecules have been

identified that stimulate IL-10-producing DCs. B. pertussis, the etiologic

agent of whooping cough, causes a severe and protracted respiratory

disease, often complicated by secondary infections that can have a lethal

outcome in young children. Recovery from infection in humans and

�207

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

mice is associated with the development of B. pertussis-specific Th1 cells

(Mills et al., 1993; Ryan et al., 1997). However, antigen-specific Th1 immune

responses in the lungs of infected mice are severely suppressed during

the acute phase of infection (McGuirk et al., 1998). The B. pertussis

virulence factors, FHA and CyaA, have been shown to inhibit IL-12 and

enhance IL-10 production from macrophages and DCs and as a result

selectively stimulate the induction of IL-10 secreting Tr1 cells from naive

Figure 10.1. Bacteria-derived immunomodulatory molecules can promote the induction

of Th1 or Treg cells by modulating DC activation. Bacteria-derived immunomodulatory

molecules, including LPS and CpG motifs in bacterial DNA, that bind to Toll-like

receptors (TLR) on dendritic cells (DCs) activate signaling pathways that enhance MHC

class II, CD80, CD86 and CD40 expression and IL-12 production by DCs, promote the

induction of Th1 cells from naive T cells. In contrast a distinct set of bacteria-derived

immunomodulatory molecules, including FHA and CyaA from B. pertussis, LAM from

M. tuberculosis and cholera toxin (CT) that bind to other PPR, including CD11b/CD18, GM1

or DC-SIGN, activate signaling pathways that enhance MHC class II and CD80 expression

and IL-10 production, but suppress CD40 expression and IL-12 production by DCs,

promote the induction of Treg cells from naive T cells. These Treg cells suppress

immune responses mediated by Th1 cells.�

208

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

Tcells (McGuirk et al., 2002; McGuirk and Mills, 2000; Boyd et al., 2005; Ross

et al., 2004). Furthermore, Tr1 clones specific for FHA have been generated

from the lungs of acutely infected mice, but could not be generated from the

spleens of infected or convalescent mice, or the lungs of naive mice (McGuirk

et al., 2002).

In a murine model of eosinophilic airway inflammation, treatment

with M. vaccae has been demonstrated to have suppressive activity on

airway eosinophilia (Zuany-Amorim et al., 2002). Treatment of mice

with killed M. vaccae suspension gives rise to CD11cþ cells that express

high levels of IL-10, TGF-b and IFN-a mRNA (Adams et al., 2004),

cytokines which are thought to strongly promote the differentiation of

Treg cells. Indeed, M. vaccae treatment has been shown to promote the

induction of allergen-specific CD4þCD45RBlow Treg cells, which not only

confer protection against airway epithelium damage but also reduce

other pathophysiological readouts such as bronchial hyperresponsiveness

(Zuany-Amorim et al., 2002).

10.6 ROLE OF INDUCIBLE AND NATURAL TREG CELLS

IN BACTERIAL INFECTIONS

There is now mounting evidence that numerous bacteria and their pro-

ducts selectively promote the differentiation of Treg as a deliberate strategy

to misdirect the immune response and thus facilitate their persistence

in vivo. As previously discussed, the respiratory pathogen B. pertussis

causes a protracted infection associated with suppressed Th1 responses

during the acute phase of infection. Tr1 clones specific for FHA and

pertactin have been generated from the lungs of acutely infected mice.

(McGuirk et al., 2002).

Treg cells may also play a significant role in the persistence of chronic

bacterial infections. In tuberculosis, 15 per cent of infected patients fail

to respond to intradermal injection with purified protein derivative (PPD)

(Boussiotis et al., 2000). T cells from patients with normal DTH

responses proliferate and secrete IFN-g. In contrast, T cells from unrespon-

sive patients proliferate poorly and secrete high levels of IL-10 (Boussiotis

et al., 2000; Delgado et al., 2002). Furthermore, recent studies in IL-10

transgenic mice demonstrate that suppression of protective Th1 responses

and increased susceptibility to reactivation of tuberculosis is strongly

influenced by the expression of IL-10 during the latent phase of infection

(Turner et al., 2002). In addition, some bacterial products have been shown to

be capable of directly inducing Treg cells. Streptococcus pyogenes, a member of

�209

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

the group A streptococci, causes numerous diseases in humans, including

pharyngitis, skin infections and post infectious rheumatic fever. One of the

major virulence factors of S. pyogenes is the M protein, which is thought

to play a role in bacterial adhesion. However, a recent study has demonstrated

that M protein interacts with CD46 on T cells and directly promotes

the differentiation of IL-10 secreting Treg cells from naive precursors

(Price et al., 2005).

Although the induction of Treg cells following infection in many

cases promotes bacterial persistence, bacterial-specific Treg cells are not

exclusively detrimental for the host. The intestine contains a large quantity

of resident bacteria that are a significant source of both antigen and

pro-inflammatory molecules. However, despite these immune responses

the intestine remains in a state of controlled inflammation, suggesting

that mucosal immune responses to enteric bacteria are tightly regulated.

Support for this concept has come from a number of experimental systems

demonstrating that altered regulation of intestinal T cell function can

result in chronic inflammatory gut disorders. In the murine model

of inflammatory bowel disease, adoptive transfer of CD4þCD45RBhigh cells

from normal mice to SCID recipients results in severe colitis, wasting

and death. However, co-transfer of the reciprocal CD4þCD45RBlow subset

prevents disease, due to the presence of Treg cells within this subset

that secrete high levels of IL-10 and TGF-b (Powrie et al., 1994). Furthermore,

it is now well established that IL-10 deficient mice not housed under germ

free or specific pathogen-free conditions develop spontaneous colitis (Powrie

et al., 1996). Therefore, one potentially important immunoregulatory

function of certain bacterial species is their involvement in the generation

of distinct T cell subtypes and maintenance of immune homeostasis.

Direct evidence for the role of bacteria-specific Treg cells in the

maintenance of homeostasis has come from recent studies on a common

gut pathogen H. hepaticus encountered in animal facilities. H. hepaticus

infection induced a population of CD4þCD45RBlow Treg cells that inhi-

bited the development of colitis triggered by this Gram-negative bacterium

in IL-10 knockout mice (Kullberg et al., 2001, 2002). The protective Treg

cells from H. hepaticus infected mice were enriched within the CD25�

CD45RBlow subset, but absent from naive mice, suggesting that these Treg

cells, rather than being endogenous, represent a memory population

resulting from previous exposure to bacterial antigen. The major question

that arises from this study is whether gut flora-specific Treg cells, similar

to those described above, prevent the appearance of inflammatory bowel

disease in humans. Evidence in support of this possibility comes from

�

210

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

studies demonstrating T cell tolerance in the intestine toward resident

gut flora is mediated by bacterial antigen-specific CD4þ T cells that

secrete IL-10 and TGF-b (Khoo et al., 1997). In addition, it has also been

suggested that suppressed CD4þ memory T cell responsiveness from

H. pylori infected individuals is due to the presence of pathogen-specific

Treg cells (Lundgren et al., 2003). It has also been suggested that the primary

role of natural CD4þCD25þ Treg cells is to limit immune mediated

pathology.

Another example of a beneficial role for regulatory cells in controlling

exaggerated inflammatory immune responses has come from studies on

infection of Toll-like receptor (TLR)-4 deficient mice with the respiratory

pathogen B. pertussis. During the acute phase of infection, Th1 responses

were shown to be enhanced and IL-10 producing T cells significantly

reduced in TLR-4 deficient mice. This was associated with enhanced

inflammatory cytokine production, cellular infiltration and severe patho-

logical changes in the lungs of the TLR-4 deficient mice (Higgins et al., 2003).

These findings suggest that ligation of conserved pathogen molecules

to PRRs promotes the induction of Treg cells that prevents immune-

mediated pathology. Therefore, IL-10 production from innate cells leading

to the differentiation of Treg cells, may represent a protective strategy

adopted by the host to limit collateral damage mediated by excessive

pathogen-stimulated inflammatory responses.

10.7 CONCLUSIONS

The immune system is capable of mounting an array of innate and

adaptive responses that can prevent or control bacterial diseases and

confer immunity to re-infection with pathogenic bacteria. However, protec-

tive immune responses, especially those against intracellular pathogens can

cause collateral damage to host tissues and must be tightly regulated

to prevent excessive immunopathology. Furthermore, pathogens have

evolved a variety of immune subversion approaches for subverting

protective immunity and prolonging their survival in the host. Bacteria

and their products can induce regulatory cytokines, especially IL-10 and

TGF-b, from innate immune cells, including macrophages and DCs,

which in turn can generate bacteria-specific Treg cells. There is growing

evidence of inducible Treg cells in bacterial infections, which either sup-

press effector immune response and thereby delay or prevent pathogen

clearance, or allow pathogen elimination with limited inflammatory

pathology. Treg cells and anti-inflammatory cytokines play a central role

�211

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

in regulating immune responses to self and foreign antigens, and a better

understanding of their role in immunity during bacterial infection should

help in the design of new or improved vaccines and therapies against

many diseases, that are still responsible for high levels of morbidity and

mortality worldwide.

REFERENCES

Adams, V. C., Hunt, J. R., Martinelli, R., Palmer, R., Rook, G. A., and Brunet, L. R.

(2004). Mycobacterium vaccae induces a population of pulmonary CD11cþ

cells with regulatory potential in allergic mice. Eur. J. Immunol. 34, 631�8.

Agrawal, A., Lingappa, J., Leppla, S. H., Agrawal, S., Jabbar, A., Quinn, C.,

and Pulendran, B. (2003). Impairment of dendritic cells and

adaptive immunity by anthrax lethal toxin. Nature 424, 329�34.

al-Ramadi, B. K., Brodkin, M. A., Mosser, D. M., and Eisenstein,

T. K. (1991a). Immunosuppression induced by attenuated Salmonella.

Evidence for mediation by macrophage precursors. J. Immunol.

146, 2737�46.

al-Ramadi, B. K., Chen, Y. W., Meissler, J. J., Jr., and Eisenstein, T. K. (1991b).

Immunosuppression induced by attenuated Salmonella. Reversal by

IL-4. J. Immunol. 147, 1954�61.

Anjuere, F., Luci, C., Lebens, M., Rousseau, D., Hervouet, C., Milon, G.,

Holmgren, J., Ardavin, C., and Czerkinsky, C. (2004). In vivo

adjuvant-induced mobilization and maturation of gut dendritic

cells after oral administration of cholera toxin. J. Immunol. 173,

5103�11.

Bagley, K. C., Abdelwahab, S. F., Tuskan, R. G., Fouts, T. R., and Lewis, G. K.

(2002a). Cholera toxin and heat-labile enterotoxin activate human

monocyte-derived dendritic cells and dominantly inhibit cytokine

production through a cyclic AMP-dependent pathway. Infect. Immun.

70, 5533�9.

Bagley, K. C., Abdelwahab, S. F., Tuskan, R. G., Fouts, T. R., and Lewis, G. K.

(2002b). Pertussis toxin and the adenylate cyclase toxin from Bordetella

pertussis activate human monocyte-derived dendritic cells and dominantly

inhibit cytokine production through a cAMP-dependent pathway. J. Leukoc.

Biol. 72, 962�9.

Bagley, K. C., Abdelwahab, S. F., Tuskan, R. G., and Lewis, G. K. (2005).

Pasteurella multocida toxin activates human monocyte-derived and

murine bone marrow-derived dendritic cells in vitro but suppresses

antibody production in vivo. Infect. Immun. 73, 413�21.

�

212

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

Balkhi, M. Y., Sinha, A., and Natarajan, K. (2004). Dominance of CD86,

transforming growth factor- beta 1, and interleukin-10 in

Mycobacterium tuberculosis secretory antigen-activated dendritic cells

regulates T helper 1 responses to mycobacterial antigens. J. Infect. Dis.

189, 1598�609.

Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y. J.,

Pulendran, B., and Palucka, K. (2000). Immunobiology of dendritic cells.

Annu. Rev. Immunol. 18, 767�811.

Barnes, P. F., Chatterjee, D., Abrams, J. S., Lu, S., Wang, E., Yamamura, M.,

Brennan, P. J., and Modlin, R. L. (1992). Cytokine production induced

by Mycobacterium tuberculosis lipoarabinomannan. Relationship to

chemical structure. J. Immunol. 149, 541�7.

Barrat, F. J., Cua, D. J., Boonstra, A., Richards, D. F., Crain, C., Savelkoul, H. F.,

de Waal-Malefyt, R., Coffman, R. L., Hawrylowicz, C. M., and O’Garra, A.

(2002). In vitro generation of interleukin 10-producing regulatory

CD4(þ) T cells is induced by immunosuppressive drugs and inhibited

by T helper type 1 (Th1)- and Th2-inducing cytokines. J. Exp. Med.

195, 603�16.

Beuscher, H. U., Rodel, F., Forsberg, A., and Rollinghoff, M. (1995). Bacterial

evasion of host immune defense: Yersinia enterocolitica encodes a suppressor

for tumor necrosis factor alpha expression. Infect. Immun. 63, 1270�7.

Boirivant, M., Fuss, I. J., Ferroni, L., De Pascale, M., and Strober, W. (2001).

Oral administration of recombinant cholera toxin subunit B inhibits

IL-12-mediated murine experimental (trinitrobenzene sulfonic acid)

colitis. J. Immunol. 166, 3522�32.

Bonecini-Almeida, M. G., Ho, J. L., Boechat, N., Huard, R. C., Chitale, S., Doo, H.,

Geng, J., Rego, L., Lazzarini, L. C., Kritski, A. L., Johnson, W. D., Jr.,

McCaffrey, T. A., and Silva, J. R. (2004). Down-modulation of lung

immune responses by interleukin-10 and transforming growth factor

beta (TGF-beta) and analysis of TGF-beta receptors I and II in active

tuberculosis. Infect. Immun. 72, 2628�34.

Boussiotis, V. A., Tsai, E. Y., Yunis, E. J., Thim, S., Delgado, J. C., Dascher, C. C.,

Berezovskaya, A., Rousset, D., Reynes, J. M., and Goldfeld, A. E. (2000).

IL-10-producing T cells suppress immune responses in anergic

tuberculosis patients. J. Clin. Invest. 105, 1317�25.

Boyd, A. P., Ross, P. J., Conroy, H., Mahon, N., Lavelle, E. C., and Mills, K. H.

(2005). Bordetella pertussis adenylate cyclase toxin modulates innate

and adaptive immune responses: distinct roles for acylation and

enzymatic activity in immunomodulation and cell death. J. Immunol.

175, 730�8.

�213

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

Braun, M. C., He, J., Wu, C. Y., and Kelsall, B. L. (1999). Cholera toxin suppresses

interleukin (IL)-12 production and IL-12 receptor beta1 and beta2 chain

expression. J. Exp. Med. 189, 541�52.

Brzoza, K. L., Rockel, A. B., and Hiltbold, E. M. (2004). Cytoplasmic entry

of Listeria monocytogenes enhances dendritic cell maturation and T cell

differentiation and function. J. Immunol. 173, 2641�51.

Byrne, P., McGuirk, P., Todryk, S., and Mills, K. H. (2004). Depletion

of NK cells results in disseminating lethal infection with Bordetella

pertussis associated with a reduction of antigen-specific Th1 and

enhancement of Th2, but not Tr1 cells. Eur. J. Immunol. 34,

2579�88.

Chen, W., Shu, D., and Chadwick, V. S. (2001). Helicobacter pylori infection:

mechanism of colonization and functional dyspepsia reduced colonization

of gastric mucosa by Helicobacter pylori in mice deficient in interleukin-10.

J. Gastroenterol. Hepatol. 16, 377�83.

Chiao, J. W., Villalon, P., Schwartz, I., and Wormser, G. P. (2000). Modulation

of lymphocyte proliferative responses by a canine Lyme disease vaccine

of recombinant outer surface protein A (OspA). FEMS Immunol. Med.

Microbiol. 28, 193�6.

Chieppa, M., Bianchi, G., Doni, A., Del Prete, A., Sironi, M., Laskarin, G.,

Monti, P., Piemonti, L., Biondi, A., Mantovani, A., Introna, M., and

Allavena, P. (2003). Cross-linking of the mannose receptor on monocyte-

derived dendritic cells activates an anti-inflammatory immunosuppressive

program. J. Immunol. 171, 4552�60.

Christensen, H. R., Frokiaer, H., and Pestka, J. J. (2002). Lactobacilli

differentially modulate expression of cytokines and maturation

surface markers in murine dendritic cells. J. Immunol. 168, 171�8.

Dahl, K. E., Shiratsuchi, H., Hamilton, B. D., Ellner, J. J., and Toossi, Z. (1996).

Selective induction of transforming growth factor beta in human monocytes

by lipoarabinomannan of Mycobacterium tuberculosis. Infect. Immun.

64, 399�405.

de Souza, M. S., Smith, A. L., Beck, D. S., Terwilliger, G. A., Fikrig, E., and

Barthold, S. W. (1993). Long-term study of cell-mediated

responses to Borrelia burgdorferi in the laboratory mouse. Infect. Immun.

61, 1814�22.

Deckert, M., Soltek, S., Geginat, G., Lutjen, S., Montesinos-Rongen, M.,

Hof, H., and Schluter, D. (2001). Endogenous interleukin-10 is required

for prevention of a hyperinflammatory intracerebral immune

response in Listeria monocytogenes meningoencephalitis. Infect. Immun.

69, 4561�71.

�

214

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

Delgado, J. C., Tsai, E. Y., Thim, S., Baena, A., Boussiotis, V. A., Reynes, J. M.,

Sath, S., Grosjean, P., Yunis, E. J., and Goldfeld, A. E. (2002).

Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary

tuberculosis patients from rural Cambodia. Proc. Natl Acad. Sci. U S A

99, 7576�81.

Demangel, C., Bertolino, P., and Britton, W. J. (2002). Autocrine IL-10

impairs dendritic cell (DC)-derived immune responses to mycobacterial

infection by suppressing DC trafficking to draining lymph nodes and

local IL-12 production. Eur. J. Immunol. 32, 994�1002.

d’Ostiani, C. F., Del Sero, G., Bacci, A., Montagnoli, C., Spreca, A., Mencacci, A.,

Ricciardi-Castagnoli, P., and Romani, L. (2000). Dendritic cells discriminate

between yeasts and hyphae of the fungus Candida albicans. Implications

for initiation of T helper cell immunity in vitro and in vivo. J. Exp. Med. 191,

1661�74.

Erfurth, S. E., Grobner, S., Kramer, U., Gunst, D. S., Soldanova, I.,

Schaller, M., Autenrieth, I. B., and Borgmann, S. (2004). Yersinia

enterocolitica induces apoptosis and inhibits surface molecule expression

and cytokine production in murine dendritic cells. Infect. Immun. 72,

7045�54.

Gagliardi, M. C., Sallusto, F., Marinaro, M., Langenkamp, A., Lanzavecchia, A.,

and De Magistris, M. T. (2000). Cholera toxin induces maturation of

human dendritic cells and licences them for Th2 priming. Eur. J. Immunol.

30, 2394�403.

Gagliardi, M. C., Teloni, R., Giannoni, F., Pardini, M., Sargentini, V., Brunori, L.,

Fattorini, L., and Nisini, R. (2005). Mycobacterium bovis Bacillus

Calmette-Guerin infects DC-SIGN-dendritic cell and causes the inhibition

of IL-12 and the enhancement of IL-10 production. J. Leukoc. Biol.

78, 106�13.

Geijtenbeek, T. B., Van Vliet, S. J., Koppel, E. A., Sanchez-Hernandez, M.,

Vandenbroucke-Grauls, C. M., Appelmelk, B., and Van Kooyk, Y. (2003).

Mycobacteria target DC-SIGN to suppress dendritic cell function. J. Exp.

Med. 197, 7�17.

Happel, K. I., Zheng, M., Young, E., Quinton, L. J., Lockhart, E.,

Ramsay, A. J., Shellito, J. E., Schurr, J. R., Bagby, G. J., Nelson, S., and

Kolls, J. K. (2003). Cutting edge: roles of Toll-like receptor 4 and IL-23

in IL-17 expression in response to Klebsiella pneumoniae infection.

J. Immunol. 170, 4432�6.

Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H.,

Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000).

A Toll-like receptor recognizes bacterial DNA. Nature 408, 740�5.

�215

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

Higgins, S. C., Lavelle, E. C., McCann, C., Keogh, B., McNeela, E., Byrne, P.,

O’Gorman, B., Jarnicki, A., McGuirk, P., and Mills, K. H. (2003). Toll-like

receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells

and confers resistance to Bordetella pertussis by inhibiting inflammatory

pathology. J. Immunol. 171, 3119�27.

Hoover, D. L., Friedlander, A. M., Rogers, L. C., Yoon, I. K., Warren, R. L.,

and Cross, A. S. (1994). Anthrax edema toxin differentially regulates

lipopolysaccharide-induced monocyte production of tumor necrosis factor

alpha and interleukin-6 by increasing intracellular cyclic AMP. Infect.

Immun. 62, 4432�9.

Hori, S. and Sakaguchi, S. (2004). Foxp3: a critical regulator of the development

and function of regulatory T cells. Microbes Infect. 6, 745�51.

Ishibashi, Y., Claus, S., and Relman, D. A. (1994). Bordetella pertussis filamentous

hemagglutinin interacts with a leukocyte signal transduction complex and

stimulates bacterial adherence to monocyte CR3 (CD11b/CD18). J. Exp.

Med. 180, 1225�33.

Jacobs, M., Brown, N., Allie, N., Gulert, R., and Ryffel, B. (2000). Increased

resistance to mycobacterial infection in the absence of interleukin-10.

Immunology 100, 494�501.

Jacobs, M., Fick, L., Allie, N., Brown, N., and Ryffel, B. (2002). Enhanced

immune response in Mycobacterium bovis bacille Calmette Guerin

(BCG)-infected IL-10-deficient mice. Clin. Chem. Lab. Med. 40, 893�902.

Janeway, C. A., Jr. and Medzhitov, R. (2002). Innate immune recognition.

Annu. Rev. Immunol. 20, 197�216.

Kalinski, P., Hilkens, C. M., Wierenga, E. A., and Kapsenberg, M. L. (1999).

T-cell priming by type-1 and type-2 polarized dendritic cells: the concept

of a third signal. Immunol. Today 20, 561�7.

Kaplan, G., Gandhi, R. R., Weinstein, D. E., Levis, W. R., Patarroyo, M. E.,

Brennan, P. J., and Cohn, Z. A. (1987).Mycobacterium leprae antigen-induced

suppression of T cell proliferation in vitro. J. Immunol. 138, 3028�34.

Khoo, U. Y., Proctor, I. E., and Macpherson, A. J. (1997). CD4þ T cell

down-regulation in human intestinal mucosa: evidence for

intestinal tolerance to luminal bacterial antigens. J. Immunol. 158,

3626�34.

Knipp, U., Birkholz, S., Kaup, W., Mahnke, K., and Opferkuch, W. (1994).

Suppression of human mononuclear cell response by Helicobacter pylori:

effects on isolated monocytes and lymphocytes. FEMS Immunol. Med.

Microbiol. 8, 157�66.

Kullberg, M. C., Jankovic, D., Gorelick, P. L., Caspar, P., Letterio, J. J.,

Cheever, A. W., and Sher, A. (2002). Bacteria-triggered CD4þ T regulatory

�

216

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

cells suppress Helicobacter hepaticus-induced colitis. J. Exp. Med.

196, 505�15.

Kullberg, M. C., Rothfuchs, A. G., Jankovic, D., Caspar, P., Wynn, T. A.,

Gorelick, P. L., Cheever, A. W., and Sher, A. (2001). Helicobacter

hepaticus-induced colitis in interleukin-10-deficient mice: cytokine

requirements for the induction and maintenance of intestinal inflammation.

Infect. Immun. 69, 4232�41.

Lanzavecchia, A. and Sallusto, F. (2001). Regulation of T cell immunity

by dendritic cells. Cell 106, 263�6.

Lavelle, E. C., McNeela, E., Armstrong, M. E., Leavy, O., Higgins, S. C., and

Mills, K. H. (2003). Cholera toxin promotes the induction of regulatory

T cells specific for bystander antigens by modulating dendritic cell

activation. J. Immunol. 171, 2384�92.

Lopes, L. M., Maroof, A., Dougan, G., and Chain, B. M. (2000). Inhibition of T-cell

response by Escherichia coli heat-labile enterotoxin-treated epithelial cells.

Infect. Immun. 68, 6891�5.

Lundgren, A., Suri-Payer, E., Enarsson, K., Svennerholm, A. M., and Lundin, B. S.

(2003).Helicobacter pylori-specific CD4þ CD25high regulatory Tcells suppress

memory T-cell responses to H. pylori in infected individuals. Infect. Immun.

71, 1755�62.

MacFarlane, A. S., Schwacha, M. G., and Eisenstein, T. K. (1999). In vivo

blockage of nitric oxide with aminoguanidine inhibits immunosuppression

induced by an attenuated strain of Salmonella typhimurium, potentiates

Salmonella infection, and inhibits macrophage and polymorphonuclear

leukocyte influx into the spleen. Infect. Immun. 67, 891�8.

Mahon, B. P., Sheahan, B. J., Griffin, F., Murphy, G., and Mills, K. H. (1997).

Atypical disease after Bordetella pertussis respiratory infection of mice with

targeted disruptions of interferon-gamma receptor or immunoglobulin

mu chain genes. J. Exp. Med. 186, 1843�51.

McBride, J. M., Jung, T., de Vries, J. E., and Aversa, G. (2002). IL-10 alters DC

function via modulation of cell surface molecules resulting in impaired

T-cell responses. Cell Immunol. 215, 162�72.

McGuirk, P., Johnson, P. A., Ryan, E. J., and Mills, K. H. (2000). Filamentous

hemagglutinin and pertussis toxin from Bordetella pertussis modulate

immune responses to unrelated antigens. J. Infect. Dis. 182, 1286�9.

McGuirk, P., Mahon, B. P., Griffin, F., and Mills, K. H. (1998).

Compartmentalization of T cell responses following respiratory infection

with Bordetella pertussis: hyporesponsiveness of lung T cells is associated

with modulated expression of the co-stimulatory molecule CD28. Eur. J.

Immunol. 28, 153�63.

�217

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

McGuirk, P., McCann, C., and Mills, K. H. (2002). Pathogen-specific T

regulatory 1 cells induced in the respiratory tract by a bacterial molecule

that stimulates interleukin 10 production by dendritic cells: a novel

strategy for evasion of protective T helper type 1 responses by Bordetella

pertussis. J. Exp. Med. 195, 221�31.

McGuirk, P. and Mills, K. H. (2000). Direct anti-inflammatory effect of a bacterial

virulence factor: IL-10-dependent suppression of IL-12 production by

filamentous hemagglutinin from Bordetella pertussis. Eur. J. Immunol.

30, 415�22.

Mehra, V., Brennan, P. J., Rada, E., Convit, J., and Bloom, B. R. (1984).

Lymphocyte suppression in leprosy induced by unique M. leprae

glycolipid. Nature 308, 194�6.

Mills, K. H. (2001). Immunity to Bordetella pertussis. Microbes Infect. 3,

655�77.

Mills, K. H. (2004). Regulatory T cells: friend or foe in immunity to

infection? Nat. Rev. Immunol. 4, 841�55.

Mills, K. H., Barnard, A., Watkins, J., and Redhead, K. (1993). Cell-mediated

immunity to Bordetella pertussis: role of Th1 cells in bacterial clearance

in a murine respiratory infection model. Infect. Immun. 61, 399�410.

Modlin, R. L. (1994). Th1�Th2 paradigm: insights from leprosy. J. Invest.

Dermatol. 102, 828�32.

Moore, K. W., de Waal Malefyt, R., Coffman, R. L., and O’Garra, A. (2001).

Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol.

19, 683�765.

Moser, M. and Murphy, K. M. (2000). Dendritic cell regulation of TH1�TH2

development. Nat. Immunol. 1, 199�205.

Moura, A. C., Modolell, M., and Mariano, M. (1997). Down-regulatory effect

of Mycobacterium leprae cell wall lipids on phagocytosis, oxidative

respiratory burst and tumour cell killing by mouse bone marrow

derived macrophages. Scand. J. Immunol. 46, 500�5.

Nakane, A., Asano, M., Sasaki, S., Nishikawa, S., Miura, T., Kohanawa, M.,

and Minagawa, T. (1996). Transforming growth factor beta is protective

in host resistance against Listeria monocytogenes infection in mice.

Infect. Immun. 64, 3901�4.

Natarajan, K., Latchumanan, V. K., Singh, B., Singh, S., and Sharma, P. (2003).

Down-regulation of T helper 1 responses to mycobacterial antigens due

to maturation of dendritic cells by 10-kDa Mycobacterium tuberculosis

secretory antigen. J. Infect. Dis. 187, 914�28.

Nigou, J., Zelle-Rieser, C., Gilleron, M., Thurnher, M., and Puzo, G. (2001).

Mannosylated lipoarabinomannans inhibit IL-12 production by human

�

218

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

dendritic cells: evidence for a negative signal delivered through the

mannose receptor. J. Immunol. 166, 7477�85.

Ostroukhova, M., Seguin-Devaux, C., Oriss, T. B., Dixon-McCarthy, B., Yang, L.,

Ameredes, B. T., Corcoran, T. E., and Ray, A. (2004). Tolerance induced

by inhaled antigen involves CD4(þ) T cells expressing membrane-bound

TGF-beta and FOXP3. J. Clin. Invest. 114, 28�38.

Othieno, C., Hirsch, C. S., Hamilton, B. D., Wilkinson, K., Ellner, J. J., and

Toossi, Z. (1999). Interaction of Mycobacterium tuberculosis-induced

transforming growth factor beta1 and interleukin-10. Infect. Immun.

67, 5730�5.

Pena, J. A., Li, S. Y., Wilson, P. H., Thibodeau, S. A., Szary, A. J., and Versalovic, J.

(2004). Genotypic and phenotypic studies of murine intestinal

lactobacilli: species differences in mice with and without colitis.

Appl. Environ. Microbiol. 70, 558�68.

Pena, J. A., Rogers, A. B., Ge, Z., Ng, V., Li, S. Y., Fox, J. G., and Versalovic, J.

(2005). Probiotic Lactobacillus spp. diminish Helicobacter hepaticus-induced

inflammatory bowel disease in interleukin-10-deficient mice. Infect. Immun.

73, 912�20.

Powrie, F. (2004). Immune regulation in the intestine: a balancing act

between effector and regulatory T cell responses. Ann. N Y Acad. Sci.

1029, 132�41.

Powrie, F., Carlino, J., Leach, M. W., Mauze, S., and Coffman, R. L. (1996).

A critical role for transforming growth factor-b but not interleukin 4

in the suppression of T helper type 1-mediated colitis by CD45RBlow

CD4þ T cells. J. Exp. Med. 183, 2669�74.

Powrie, F., Correa-Oliveira, R., Mauze, S., and Coffman, R. L. (1994).

Regulatory interactions between CD45RBhigh and CD45RBlow CD4þ

T cells are important for the balance between protective and pathogenic

cell-mediated immunity. J. Exp. Med. 179, 589�600.

Price, J. D., Schaumburg, J., Sandin, C., Atkinson, J. P., Lindahl, G., and

Kemper, C. (2005). Induction of a regulatory phenotype in human

CD4þ T cells by streptococcal M protein. J. Immunol. 175, 677�84.

Redpath, S., Ghazal, P., and Gascoigne, N. R. (2001). Hijacking and exploitation

of IL-10 by intracellular pathogens. Trends. Microbiol. 9, 86�92.

Reis e Sousa, C. (2004). Activation of dendritic cells: translating innate into

adaptive immunity. Curr. Opin. Immunol. 16, 21�5.

Rosenfeldt, V., Benfeldt, E., Nielsen, S. D., Michaelsen, K. F., Jeppesen, D. L.,

Valerius, N. H., and Paerregaard, A. (2003). Effect of probiotic Lactobacillus

strains in children with atopic dermatitis. J. Allergy Clin. Immunol. 111,

389�95.

�219

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

Ross, P. J., Lavelle, E. C., Mills, K. H., and Boyd, A. P. (2004). Adenylate

cyclase toxin from Bordetella pertussis synergizes with lipopolysaccharide

to promote innate interleukin-10 production and enhances the induction

of Th2 and regulatory T cells. Infect. Immun. 72, 1568�79.

Ryan, E. J., McNeela, E., Pizza, M., Rappuoli, R., O’Neill, L., and Mills, K. H.

(2000). Modulation of innate and acquired immune responses by Escherichia

coli heat-labile toxin: distinct pro- and anti-inflammatory effects of the

nontoxic AB complex and the enzyme activity. J. Immunol. 165, 5750�9.

Ryan, M., Murphy, G., Gothefors, L., Nilsson, L., Storsaeter, J., and Mills, K. H.

(1997). Bordetella pertussis respiratory infection in children is associated

with preferential activation of type 1 T helper cells. J. Infect. Dis. 175,

1246�50.

Sakaguchi, S. (2000). Regulatory T cells: key controllers of immunologic

self-tolerance. Cell 101, 455�8.

Salgame, P. R., Birdi, T. J., Lad, S. J., Mahadevan, P. R., and Antia, N. H.

(1984). Mechanism of immunosuppression in leprosy � macrophage

membrane alterations. J. Clin. Lab. Immunol. 14, 145�9.

Schaible, U. E., and Kaufmann, S. H. (2000). CD1 and CD1-restricted T cells

in infections with intracellular bacteria. Trends Microbiol. 8, 419�25.

Schoppet, M., Bubert, A., and Huppertz, H. I. (2000). Dendritic cell function

is perturbed by Yersinia enterocolitica infection in vitro. Clin. Exp. Immunol.

122, 316�23.

Sewnath, M. E., Olszyna, D. P., Birjmohun, R., ten Kate, F. J., Gouma, D. J., and

van Der Poll, T. (2001). IL-10-deficient mice demonstrate multiple organ

failure and increased mortality during Escherichia coli peritonitis

despite an accelerated bacterial clearance. J. Immunol. 166, 6323�31.

Shannon, J. G., Howe, D., and Heinzen, R. A. (2005). Virulent Coxiella burnetii

does not activate human dendritic cells: role of lipopolysaccharide as a

shielding molecule. Proc. Natl Acad. Sci. U S A 102, 8722�7.

Sing, A., Roggenkamp, A., Geiger, A. M., and Heesemann, J. (2002). Yersinia

enterocolitica evasion of the host innate immune response by V

antigen-induced IL-10 production of macrophages is abrogated in

IL-10-deficient mice. J. Immunol. 168, 1315�21.

Smits, H. H., Engering, A., van der Kleij, D., de Jong, E. C., Schipper, K.,

van Capel, T. M., Zaat, B. A., Yazdanbakhsh, M., Wierenga, E. A.,

van Kooyk, Y., and Kapsenberg, M. L. (2005). Selective probiotic bacteria

induce IL-10-producing regulatory T cells in vitro by modulating

dendritic cell function through dendritic cell-specific intercellular

adhesion molecule 3-grabbing nonintegrin. J. Allergy Clin. Immunol.

115, 1260�7.

�

220

M.

T.

BR

AD

Y,

P.

MC

GU

IRK

AN

DK

.H

.G

.M

ILL

S

Tournier, J. N., Quesnel-Hellmann, A., Mathieu, J., Montecucco, C., Tang, W. J.,

Mock, M., Vidal, D. R., and Goossens, P. L. (2005). Anthrax edema toxin

cooperates with lethal toxin to impair cytokine secretion during infection

of dendritic cells. J. Immunol. 174, 4934�41.

Turner, J., Gonzalez-Juarrero, M., Ellis, D. L., Basaraba, R. J., Kipnis, A.,

Orme, I. M., and Cooper, A. M. (2002). In vivo IL-10 production reactivates

chronic pulmonary tuberculosis in C57BL/6 mice. J. Immunol.

169, 6343�51.

van Kooyk, Y. and Geijtenbeek, T. B. (2003). DC-SIGN: escape mechanism

for pathogens. Nat. Rev. Immunol. 3, 697�709.

Yao, T., Mecsas, J., Healy, J. I., Falkow, S., and Chien, Y. (1999). Suppression

of T and B lymphocyte activation by a Yersinia pseudotuberculosis

virulence factor, yopH. J. Exp. Med. 190, 1343�50.

Zeidner, N., Mbow, M. L., Dolan, M., Massung, R., Baca, E., and Piesman, J.

(1997). Effects of Ixodes scapularis and Borrelia burgdorferi on modulation

of the host immune response: induction of a TH2 cytokine response

in Lyme disease-susceptible (C3H/HeJ) mice but not in

disease-resistant (BALB/c) mice. Infect. Immun. 65, 3100�6.

Zuany-Amorim, C., Sawicka, E., Manlius, C., Le Moine, A., Brunet, L. R.,

Kemeny, D. M., Bowen, G., Rook, G., and Walker, C. (2002). Suppression

of airway eosinophilia by killed Mycobacterium vaccae-induced

allergen-specific regulatory T-cells. Nat. Med. 8, 625�9.

�221

SU

PP

RE

SS

ION

OF

IMM

UN

ER

ES

PO

NS

ES

BY

BA

CT

ER

IA

CHAPTER 11

Dendritic cells in the gut and their possible rolein disease

Christoph BeckerUniversity of Mainz

The gut represents the largest lymphoid tissue of the whole body. The

delicate task of the intestinal immune system is the discrimination of

harmless food antigens and the commensal bacterial flora from harmful

pathogens. Under normal physiologic conditions, immune tolerance is

induced to non-pathogenic stimuli while effective immune responses

are generated toward dangerous pathogens. Thus ‘‘decision making’’ is an

important feature of the intestinal immune system. If inappropriate

responses are generated, serious inflammation of the small and large

intestine may develop. Crohn’s disease (CD) and ulcerative colitis are the

two prototypes of such inflammatory bowel disease that are believed to

develop as a consequence of a disregulated immune response toward

harmless antigens. Despite our limited knowledge on the mechanisms of

such ‘‘decision making’’ in the gut, recent evidence suggest an important

role of intestinal dendritic cells1. Dendritic cells (DCs) can be found in

large numbers throughout the gastrointestinal tract where they usually

build a tight network underlying the epithelium1,2. This chapter will discuss

their contribution to the induction of tolerance and immunity in the

intestinal immune system as well as a possible role of these DCs in localized

immune responses predisposing the terminal ileum for the development

of inflammatory bowel disease (IBD).

11.1 DENDRITIC CELLS IN THE INTESTINAL IMMUNE SYSTEM:

AN OVERVIEW

The intestinal immune system can be functionally separated into an

inductive site and an effector site1. The prototypic inductive site in

the small intestine is the Peyer’s patch, a localized lymphoid structure

placed within the bowel wall. Peyer’s patches are composed of a

specialized, so-called follicle-associated epithelium, the adjacent so-called

subepithelial dome, B cell follicles containing germinal centers and

surrounding interfollicular regions. Antigens and microorganisms are

thought to be transported from the gut lumen via M-cells, specialized

epithelial cells within the follicle-associated epithelium, into the Peyer’s

patch follicle. In the subepithelial dome region they get taken up by

local dendritic cells. Dendritic cells in the subepithelial dome represent an

immature phenotype. However, upon antigen encounter, they are thought

to mature and migrate into the T cell rich area of the interfollicular

regions. Here the dendritic cells display a mature phenotype with

high expression of the costimulatory molecules CD80 and CD86 thus

representing potent antigen-presenting cells, ready to stimulate resident

naıve T cells. Stimulated T cells can then circulate and migrate into the

lamina propria which is the main effector site of the intestinal immune

system.

Dendritic cells in the Peyer’s patches like elsewhere in the body com-

monly express the integrin CD11c. However, three distinct subpopulations

of dendritic cells have been identified in murine Peyer’s patches based

on their expression of the molecules CD11b and CD8a: CD11cþ CD11bþ

CD8a� myeloid dendritic cells, CD11cþ CD11b� CD8aþ lymphoid dendritic

cells, CD11cþ CD11b� CD8a� double negative (DN) dendritic cells3.

Interestingly these subpopulations are not evenly distributed within

the Peyer’s patch; they rather reside in distinct areas3,4. While the myeloid

CD11bþ DCs were found almost exclusively in the subepithelial dome,

lymphoid CD8aþ DCs were found in the interfollicular regions. Third,

double negative DCs were found at both locations and in addition within

the follicle-associated epithelium3,5. Despite the fact that most studies

on the role of DCs in the gut were performed with cells isolated from

Peyer’s patches or mesenteric lymph nodes, DCs are also prominent in

the mucosa1. High numbers of DCs form a close meshed network within

the lamina propria of the small intestine, especially within the villi. In

contrast to the small intestine, the colon has no Peyer’s patches, but houses

numerous isolated lymphoid follicles with some structural similarities

to Peyer’s patches implicating that they are also involved in inducing

immune responses. In addition aggregates of DCs can also be found in

the lamina propria of the colon, although at lower numbers than in the

small intestine.

�

224

C.

BE

CK

ER

11.1.1 Tolerance induction by intestinal dendritic cells

Although the intestinal immune system has to respond to pathogenic

infections with the induction of an appropriate cell-mediated immune

response, the primary task of the mucosal immune system is the induction

of tolerance. Their strategic localization directly underneath the intestinal

epithelium or the follicle-associated epithelium enables DCs to immediately

respond to the uptake of antigens. Our knowledge of the mechanisms

of tolerance induction is very limited, but there is compelling evidence

for a tolerizing function of intestinal DCs5–8. Accordingly it has been

shown, that DCs from Peyer’s patches or the lamina propria have a tendency

to induce T helper cell 2 (TH2) type immune responses and to express

anti-inflammatory cytokines like IL-10 and TGF-b5,8–10. Such TH2-like

cells express IL-6 which together with TGF-b can promote the production

of immunoglobulin A (IgA) by B cells in the follicles of Peyer’s patches11.

More recent data suggests that epithelial cells can educate DCs to

induce such TH2-like immune responses by T cells8. Thymic stromal

lymphopoietin was found to be constitutively released by epithelial cells

leading to a conditioning of colon DCs to induce TH2 rather than TH1

immune responses. A second mechanism by which mucosal DCs could

induce tolerance is the induction of regulatory T cells (Tregs). It has been

demonstrated, that plasmacytoid DCs (CD8aþ CD11cLO) can induce IL-10

producing regulatory T cells7. In another study, antigen-specific CD4þ

CD25þ regulatory T cells were found to be induced in Peyer’s patches

upon oral administration of protein antigen12,13. Recently, TGF-b was

found to induce regulatory T cells from naıve CD4þ CD25� cells in the

periphery14,15. TGF-b seems to act via an induction of FOXP3, the master-

regulator of regulatory T cell commitment in naturally occurring (thymus

derived) Treg. Since TGF-b is highly expressed in the Peyer’s patches

within 6 h after oral administration of antigen16, it is tempting to speculate

that such TGF-b released from cells including DCs in Peyer’s patches

induces regulatory T cells which then recirculate and migrate to the

mucosal effector sites in order to induce tolerance against this antigen.

11.1.2 Routes of antigen uptake in the mucosal

immune system

To participate in the induction of tolerance and immunity in the gut,

lamina propria dendritic cells have to get into contact with antigens

�225

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

present in the lumen of the gut. As mentioned above, M cells located in

the follicle-associated epithelium of Peyer’s patches are thought to be the

most important sites for the sampling of antigens from the gut lumen.

However, recent evidence question the necessity of Peyer’s patches and

support a role for alternative gateways. Accordingly, cells with features

of M cells of Peyer’s patches have been described in the (non-follicle-

associated) epithelial cell layer of intestinal villi17. Interestingly, such

villous M cells were found not only in wildtype mice, but also in mice

genetically deficient for Peyer’s patches, indicating that they develop

independent from Peyer’s patches. Functionally it was shown that villous

M cells were capable of translocating bacteria like Salmonella and

Yersinia17. Dendritic cells can also directly sample bacteria from the gut

lumen: it has been impressively demonstrated that lamina propria DCs

can extend their dendrites through the epithelial cell layer into the lumen

in order to directly sample bacterial antigens out of the lumen18.

Interestingly these DCs express tight junction proteins like occludin,

claudin-1 and zonula occludens-1 and can thereby prevent the disruption

of the epithelial barrier. The capability of DCs to directly access the lumen

and sample bacteria may provide an important function in the probing

for the presence of pathogenic microorganisms in the gut lumen. To date

it is not clear whether DCs extend their dendrites constitutively or

whether this is an induced mechanism in response to certain bacteria.

The observation that such dendrite forming DCs are more prominent in

the terminal ileum than in more proximal regions may support the latter

hypothesis, given that there is a gradient of bacteria toward the terminal

ileum of the small intestine19. The formation of such transepithelial

dendrites by lamina propria DCs was found to depend on the chemokine

receptor CX3CR1, also called fractalkine19. CX3CR1 was found to be

important for the clearance of gut-invasive pathogens by DCs.

An additional route of antigen uptake in the intestine may be the

transport of apoptotic epithelial cells to the mesenteric lymph nodes by

lamina propria DCs which occurs in the steady-state20. Such a continuous

circulation of DCs from the lamina propria to the lymphatic system has

been proposed as a mechanism by which tolerance to normal antigens in

the gut is conferred. The constitutive migration of DCs from the intestinal

mucosa to lymphatic vessels could be experimentally accelerated by a

systemic application of inflammatory cytokines or bacterial products21,22.

The underlying mechanism leading to such an induced and guided move-

ment of DCs in the gut may be the coordinated regulation of the expression

of chemokines and their receptors.

�

226

C.

BE

CK

ER

11.2 DENDRITIC CELLS IN THE PATHOGENESIS OF

INFLAMMATORY BOWEL DISEASE

Inflammatory bowel disease (IBD) comprises Crohn’s disease and ulcerative

colitis, the two major chronic inflammatory diseases of the intestine.

The cause of IBD is believed to be multifactorial23. It seems that

environmental as well as genetic factors in combination with the microbial

intestinal flora and a disregulated immune response trigger the development

of chronic inflammation of the intestine. As described above, the immune

system of the gastrointestinal tract is sensitively balanced. The processes

that lead to a loss of this balance are poorly understood. If however toler-

ance against food antigens or the commensal flora is lost, a deleterious

process is evoked, leading to a disregulated activation of mucosal immune

cells such as DCs and T cells.

11.2.1 Dendritic cells play a major role in the pathogenesis

of colitis

Increasing evidence suggests that intestinal bacterial flora is involved

in the pathogenesis of IBD24,25. Despite considerable effort, to date no

specific bacterial pathogen has been identified, implicating that different

bacteria are involved in the initiation of the pathogenic immune

response26,27. Recent data suggest that DCs in the intestine are the link

between the bacterial flora and the persistent inflammation in the colon as

seen in patients with IBD, implicating that a disregulated handling of

such bacteria play a major role in the early events that lead to the develop-

ment of chronic inflammatory and autoimmune diseases. Recently, it

was found that a subset of Crohn’s disease (CD) patients have mutations

in the gene that encodes the nucleotide-binding oligomerization domain 2

(NOD2) protein28. NOD2 has been described as a pathogen-recognition

receptor that recognizes muramyl dipeptide derived from bacterial pepti-

doglycans29. In another study, NOD2 deficiency in mice or the presence

of a CD-like NOD2 mutation was shown to increase Toll-like receptor

2-mediated activation of NF-kappaB and also TH1 responses30. Thus, NOD2

mutations may lead to disease by causing excessive TH1 responses,

suggesting that alterations in the recognition of intestinal bacteria can

contribute to IBD.

Numerous mouse models of IBD have been developed. From all these

models, the so-called adoptive transfer model has drawn much attention in

recent times31,32. This model relies on the adoptive transfer of colitogenic

�227

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

CD4þCD45RBHI Tcells into SCID recipient mice. SCID mice have a mutant

phenotype and as a result of this do not develop functional Tcells and B cells.

The transferred colitogenic T cells repopulate the SCID hosts and lead to

a severe chronic colitis after 8 to 12 weeks. Importantly the colon pathology

very much resembles that seen in patients with CD, making it an important

model for the investigation of human IBD31. Studies performed with this

model implicated an important role for DCs in the early events of colitis

manifestation. Transferred T cells were demonstrated to form aggregates

with CD11cþ DC in the lamina propria, where they substantially prolifer-

ated33. Interestingly, the authors of this report observed a delay between the

influx of T cells into the mesenteric lymph nodes and the formation of

aggregates between T cells and DCs in the lamina propria. The authors

hypothesized that after adoptive transfer, T cells went to the mesenteric

lymph nodes to be primed by DCs before they finally migrated to the lamina

propria to fulfill their effector function. Other studies also demonstrated that

a priming of the Tcells by DCs was necessary for the development of colitis in

the adoptive transfer model34. In this study, the priming of colitogenic Tcells

was shown to be dependent on OX40L since blocking the interaction of

OX40L present on T cells with OX40 present on DCs protected mice from

colitis development. Consequently high numbers of OX40L expressing

CD11cþ CD11bþ CD8a� DCs were found in mice with colitis. Thus it seems

that after adoptive transfer but before the onset of disease, T cells are primed

by a direct interaction with DCs in the mesenteric lymph nodes and

subsequently migrate into the lamina propria where they undergo a massive

proliferation which then finally leads to a chronic colitis characterized by the

production of vast amount of inflammatory cytokines. Not only do T cells

proliferate in the course of disease development, but also the expansion of

DCs in the lamina propria of colitis bearing animals has been described35.

These DCs were shown to display an upregulation of costimulatory

molecules like CD80, CD86 and CD40. Therefore, it seems that T cells and

DCs in the lamina propria support each other’s growth and stimu-

lation, thereby sustaining the inflammation.

11.2.2 Dendritic cells polarize T helper cell responses leading

to Crohn’s disease

Despite a general role of DCs in the priming of T cells and the induction of

colitis, DCs could also specifically direct the kind of T helper cell response

generated in IBD patients. Recent evidence suggest that in CD, T cells

represent a dominant TH1 phenotype while in ulcerative colitis T cells are

�

228

C.

BE

CK

ER

predominantly skewed toward a TH2-like phenotype36. When CD patients

were compared to control patients, isolated lamina propria CD4þ T cells

from CD samples produced elevated levels of the TH1 cytokine IFN-g while

the expression of the TH2 cytokines IL-4 and IL-5 were diminished. In line

with such a polarized model, IL-12 produced by activated DCs has been

proposed as a key cytokine in the pathogenesis of CD but not in the

pathogenesis of ulcerative colitis37–39. IL-12 (p40/p35) is rapidly released

from DCs after stimulation via Toll-like receptors and studies in mouse

models of CD have reported an essential role of IL-12 in the pathogenesis of

chronic intestinal inflammation. Neutralizing IL-12 p40 in these animals

using a specific antibody resulted in the protection from IBD39–41. Further

studies revealed that mice transgenic for STAT-4, a signaling molecule

downstream of the IL-12 receptor, are highly suscep-tible to TH1 driven

colitis42. Antibodies against IL-12 p40 are currently in clinical trials. A recent

phase-2 study reported long-lasting remission in 50 per cent of patients

treated43. Thus IL-12 is a promising target for future therapeutic intervention

in patients with CD. However, many of the investigations regarding

IL-12 were performed using antibodies against the IL-12 p40 subunit.

Figure 11.1. Model of constitutive IL-23 production in the terminal ileum: dendritic

cells (DCs) sample bacteria from the lumen and produce IL-23. IL-23 in turn can

stimulate infiltrating memory T cells to proliferate and produce cytokines, including IL-17.

�229

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

Since the discovery that in DCs IL-12 p40 can also form heterodimers with

the recently discovered molecule p19 resulting in IL-2344, these data have to

be interpreted with care. Studies using IL-23 (p19) knockout mice in colitis

are still missing, but the discovery that in collagen-induced arthritis and in

experimental autoimmune encephalomyelitis, two commonly used models

of chronic inflammation and autoimmunity, IL-23 knockout mice but not

IL-12 knockout mice are protected from disease, implicate an important role

for DC derived IL-23 also in intestinal immune pathology45,46, Support for

a role of IL-23 in IBD comes from a recent study using mice transgenic for

IL-23. Transgenic mice constitutively overexpressing IL-23 p19 developed

a spontaneous severe inflammation in multiple organs including the gut,

with elevated levels of proinflammatory cytokines47. In another study, it

was demonstrated that IL-23 p19 was significantly increased in the inflamed

mucosa of patients with CD and to a lesser extent in patients with ulcerative

colitis as compared to patients without IBD48,49. Moreover in these studies,

elevation of IL-23 p19 levels in CD correlated with the severity of the lesions

observed during endoscopy. In a similar study, IL-17, a cytokine induced

in T cells upon stimulation with IL-23, was found to be highly expressed

in the serum and in the mucosa of patients with IBD, but not in control

patients50,51. Interestingly IL-17 was increased not only in CD patients

but also in ulcerative colitis patients, implicating an alternative route

of IL-17 induction not dependent on IL-23 in ulcerative colitis pathogenesis.

Given what is known from studies in other model systems, DC derived IL-12

may be an important pathogenic factor in the onset of IBD, while later

on, DC expressed IL-23 may drive strong memory T cell responses leading

to the perpetuation of IBD. In line with this model, IL-23 has been shown

to stimulate naıve murine T cells only poorly, an observation that is

resembled by the low expression of IL-23R on the surface of the latter

cells52. In contrast, activated memory T cells respond strongly to IL-23

but only poorly to IL-12, a finding that is paralleled by the high expression

of the IL-23R on the surface of memory T cells45,46. IL-23 has therefore

been proposed to be a late stage effector cytokine rather than a cytokine

inducing T cell commitment into a certain lineage.

However, this concept is challenged by recent reports, which have

demonstrated that IL-23 stimulation can lead to the generation of

an alternative T helper cell subset characterized by the expression of high

levels of the proinflammatory cytokine IL-17 but only low amounts of

IFN-g46,53–56. This novel T cell population was denoted THIL-17 and is

currently under investigation. A recent report by Langrish and colleagues

highlights the role of such THIL-17 cells in an animal model of EAE56.

�

230

C.

BE

CK

ER

In this report the authors demonstrate that IL-23 dependent THIL-17

cells drive autoimmune inflammation in the brain and that neutrali-

zation of soluble IL-17 by using antibodies partially protected mice from

EAE. In the animal model of collagen-induced arthritis, resistance of IL-23

knockout mice was found to depend on the absence of IL-17 producing

T helper cells but not on an impaired TH1 immune response, highlighting

again the possible role of THIL-17 cells for chronic and autoimmune

inflammation46. Both studies demonstrated higher disease levels in IL-12

(p35) knockout mice when compared to wild-type mice. The authors

attributed this observation to the higher level of IL-17 producing T helper

cells in the absence of IFN-g producing cells. Thus IL-23 induced IL-17

expression emerges as an important pathway in the pathogenesis of chronic

inflammatory and autoimmune diseases including inflammatory bowel

disease. IL-17 has furthermore been described as a cytokine that drives

proliferation, maturation and chemotaxis of neutrophils57. Beyond its

role on neutrophils, IL-17 has been shown to induce the expression of

other proinflammatory cytokines like TNF-a, IL-1 and IL-6 in various cell

types58–60. IL-17 has also been demonstrated to drive the maturation of

dendritic cells61.

However to date it is unclear whether IL-23 alone or in combination

with other stimuli can induce the generation of IL-17 producing T cells

directly from naıve T cells or whether they represent a population that has

been generated from previously activated T helper cells or early activated

T helper cells before terminal commitment. Another possibility would be

that these cells develop from a subset of TH1 cells. In support of the latter

theory, a recent report by Wu and colleagues has demonstrated the existence

of a novel IFN-g negative TH1 cell population62. In their model, IFN-gproducing TH1 cells showed immediate effector function and were short

lived, while the alternative IFN-g negative population was long-lived and

displayed memory function. It remains to be investigated whether the cell

population described in this paper resembles cells that can develop toward

the THIL-17 phenotype.

This view is also supported by studies demonstrating that IL-12 (p40)

is sustaining chronic inflammation in the colon of IL-10 knockout mice

while IFN-g is dispensable41. While other studies reported a crucial role

of IFN-g for the pathogenesis of IBD63,64, it was shown in an adoptive

transfer model of colitis, that CD45RBHi cells from IFN-g knockout mice

were still able to induce colitis, although with less severity and with a later

onset than wild-type CD45RBHi cells65. Thus it seems that IBD can

develop independently from IFN-g. Furthermore, antibodies neutralizing

�231

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

IFN-g were only able to suppress colitis when given before the onset

of colitis but not in established colitis66. Thus although IFN-g is important

in the initial events leading to IBD, it may be dispensable in established

colitis.

The third member of IL-12 related cytokines produced by activated DCs

is IL-27. IL-27 is a heterodimer composed of the two subunits EBI3

and p2867. IL-27 has been shown to cooperate with IL-12 in the priming

of naıve T cells and has also been shown to play an important role in the

pathogenesis of IBD68–70.

In summary, in CD, activated DCs in the gut seem to drive inflammation

through the release of high levels of inflammatory cytokines, especially IL-12

and the IL-12 related cytokines IL-23 and IL-27. It has to be mentioned

that beside IL-12 related cytokines, other DC derived cytokines like IL-6

and TNF-a have been shown to play an important role in the pathogenesis,

especially in the perpetuation of IBD as well71,72. These findings implicate

that in the onset of disease, DC expressed IL-12 and IL-27 may drive the

generation of a TH1 response while in later stages the disease is perpetuated

by other DC derived cytokines like IL-23, IL-6 and TNF-a. Therefore, the

important role of the above mentioned cytokines highlights the role of DCs

in the pathogenesis of IBD. In the case of TNF-a, monoclonal antibodies

targeting this cytokine have even made their way from bench to bedside

and the beneficial use of this antibody in patients with CD can be seen as a

success story of targeting dendritic cells and their mediators in disease.

11.3 A ROLE FOR LOCAL DENDRITIC CELLS IN THE

PREDISPOSITION OF THE TERMINAL ILEUM FOR COLITIS

Inflammatory bowel diseases do not affect all segments of the intestine

evenly25. Crohn’s disease may exclusively affect the small bowel, especially

the terminal ileum, which is the most common location, or it may involve

the small bowel and the colon. In contrast, in some patients the involve-

ment is limited to the colon. In more rare cases even other segments of

the gastrointestinal tract like the stomach, duodenum or the esophagus

are affected by CD. Ulcerative colitis most frequently develops in the

distal colon and progressively involves the whole colon up to the ileal

junction. However, ulcerative colitis is limited to the colon and does not

affect the small intestine. Experimental models for IBD in the mouse

most frequently affect the colon and histologic signs of colitis are most

severe in the distal colon73. In the mouse system only few models exist

in which the small intestine is involved. However, it has to be mentioned

�

232

C.

BE

CK

ER

that some frequently used experimental colitis models are based on the

rectal administration of reagents like TNBS, oxazolone or ethanol which

induce inflammation only in the contact area of the colon.

The observation that in mice and men distinct regions of the small

and large intestine are more or less prone to develop inflammation

implicates that the small and large intestine immunologically cannot be

considered a homogenous organ. The anatomical architecture in different

parts of the intestine may directly reflect its functional role in digestion and

host defense1. In agreement with such a model, differences have been

described not only in the intestinal epithelial architecture, but also in the

distribution and density of cells that are associated with the innate and

adaptive immune response in the gut, such as DCs, T and B lymphocytes

and Paneth cells. In addition, lymphoid structures like Peyer’s patches

or isolated lymphoid follicles are not evenly distributed throughout the

intestine. Since different strains of bacteria colonize distinct regions of

the intestine and since there are differences in bacterial concentrations

throughout the gut24, it is likely that the lymphoid tissue throughout the

intestine is highly adapted to the environment and plays an important role

in mediating localized immune responses to bacteria that populate the

respective compartments. The development of such a localized immune

system may therefore only be in part genetically determined. In fact, recent

evidence suggests that it is functionally dependent on the presence of the

bacterial microflora74. Interestingly, in HLA-B27 transgenic rats, the severity

of mucosal inflammation is determined by the bacterial load in the

cecum75,76. The development of colitis in T cell receptor mutant mice can

be prevented by removal of parts of the cecum77. In addition, differential

complementary determining region-3 T cell receptor usage among T cells

in different regions of the colon provides further support for localized

immune responses within the large intestine78. Recently it was demon-

strated that antigen processing by 20S proteasomes is more effective in the

small bowel as compared to the colon and other organs79. These results

imply that the manipulation of bacteria or the corresponding mucosa-

associated lymphoid tissue in specific compartments of the gut can influence

the induction of intestinal immunopathology.

Further evidence for localized immune responses in the gut comes from

a study which recently demonstrated that dendritic cells in the lamina

propria of the terminal ileum are constitutively activated by luminal

bacterial antigens, giving rise to a constitutive presence of interleukin-23

only in this part of the murine gut80. In this study, a subset of lamina propria

DCs was identified displaying constitutive IL-12 p40 promoter activity

�233

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

and IL-23 expression in the terminal ileum. Constitutively activated LPDC in

this study were demonstrated to be largely CD8a and CD11b double-negative

DCs and thus reminiscent of a DC subset recently characterized

in murine Peyer’s patches that produces high levels of IL-12 in response

to Staphylococcus aureus antigen and IFN-gamma stimulation5.

The above study raised the question about the cause of a localized

response as indicated by the difference in IL-12 p40 and IL-23 production

between LPDC from the proximal and distal small intestine of healthy

mice. One possible explanation may be that LPDC could be differentially

activated through the local microenvironment. In agreement with this

hypothesis, the authors demonstrated localization of bacteria directly in

p40 expressing cells and endocytosis of bacteria by such LPDC in the crypts

of the terminal ileum but not the proximal parts of the small bowel.

These differences might be due to an increased bacterial load of the terminal

ileum as compared to the proximal small bowel or to local changes in the

composition of the microflora. It is known that there is a general gradient

of bacterial concentration increasing toward the terminal ileum where

in CD the highest incidence of lesions can be found. Consistent with

a dominant role for resident bacteria, no increase in constitutive p40

expression was observed in the terminal ileum of mice raised under germ-

free conditions. Thus, this study implicates important functional differ-

ences between the mucosal immune systems of the colon and the proximal

and distal small intestine and provided evidence for localized DC driven

immune responses in the gut. Interestingly, an uptake of bacteria by LPDC

of the terminal ileum was only seen in the crypts where the earliest

pathological manifestations of ileitis in CD are known to occur. Based on

the above findings, it is tempting to speculate, that the elevation of IL-23

expression selectively in the ileum reflects an increased susceptibility to

inflammation in the terminal ileum, a site that is most frequently affected

in patients with CD25.

Since the functional IL-23 receptor is expressed mainly on memory

T cells and T cells in the intestinal mucosa largely represent a memory

phenotype, memory T cells migrating into the terminal ileum may be highly

susceptible to locally elevated levels of IL-23. Therefore infiltrating T cells

may develop toward inflammatory THIL-17 cells promoting inflammation

in the terminal ileum but not in other segments of the intestine. Despite

these findings, localized innate and adaptive mucosal immune responses

may also be protective for the intestine, providing an efficient response to

the respective flora while restricting extensive immunopathology.

�

234

C.

BE

CK

ER

REFERENCES

1. Mowat, A. M. (2003). Anatomical basis of tolerance and immunity to

intestinal antigens. Nat. Rev. Immunol. 3, 331–41.

2. Bilsborough, J. and Viney, J. L. (2004). Gastrointestinal dendritic cells play

a role in immunity, tolerance, and disease. Gastroenterology 127, 300–9.

3. Iwasaki, A. and Kelsall, B. L. (2000). Localization of distinct Peyer’s patch

dendritic cell subsets and their recruitment by chemokines macrophage

inflammatory protein (Mip)-3alpha, Mip-3beta, and secondary lymphoid

organ chemokine. J. Exp. Med. 191, 1381–94.

4. Kelsall, B. L. and Strober, W. (1996). Distinct populations of dendritic

cells are present in the subepithelial dome and Tcell regions of the murine

Peyer’s patch. J. Exp. Med. 183, 237–47.

5. Iwasaki, A., and Kelsall, B. L. (2001). Unique functions of Cd11bþ,

Cd8 alphaþ, and double-negative Peyer’s patch dendritic cells.

J. Immunol. 166, 4884–90.

6. Macpherson, A. J and Uhr, T. (2004). Induction of protective Iga by

intestinal dendritic cells carrying commensal bacteria. Science 303, 1662–5.

7. Bilsborough, J., George, T. C., Norment, A., and Viney, J. L. (2003). Mucosal

Cd8alphaþ Dc, with a plasmacytoid phenotype, induce differentiation and

support function of T cells with regulatory properties. Immunology 108,

481–92.

8. Rimoldi, M., Chieppa, M., Salucci, V., Avogadri, F., Sonzogni, A.,

Sampietro, G. M., Nespoli, A., Viale, G., Allavena, P., and Rescigno, M.

(2005). Intestinal immune homeostasis is regulated by the crosstalk

between epithelial cells and dendritic cells. Nat. Immunol. 6, 507–14.

9. Akbari, O., DeKruyff, R. H., and Umetsu, D. T. (2001). Pulmonary dendritic

cells producing Il-10 mediate tolerance induced by respiratory exposure

to antigen. Nat. Immunol. 2, 725–31.

10. Alpan, O., Rudomen, G., and Matzinger, P. (2001). The role of dendritic

cells, B cells, and M cells in gut-oriented immune responses. J. Immunol.

166, 4843–52.

11. Sato, A., Hashiguchi, M., Toda, E., Iwasaki, A., Hachimura, S., and

Kaminogawa, S. (2003). Cd11bþ Peyer’s patch dendritic cells secrete Il-6

and induce Iga secretion from naive B cells. J. Immunol. 171, 3684–90.

12. Tsuji, N. M., Mizumachi, K., and Kurisaki, J. (2003). Antigen-specific,

Cd4þCd25þ regulatory T cell clones induced in Peyer’s patches.

Int. Immunol. 15, 525–34.

13. Hauet-Broere, F., Unger, W. W., Garssen, J., Hoijer, M. A., Kraal, G., and

Samsom, J. N. (2003). Functional Cd25� and Cd25þ mucosal regulatory

�235

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

T cells are induced in gut-draining lymphoid tissue within 48 h after

oral antigen application. Eur. J. Immunol. 33, 2801–10.

14. Chen, W., Jin, W., Hardegen, N., Lei, K. J., Li, L., Marinos, N., McGrady, G.,

and Wahl, S. M. (2003). Conversion of peripheral Cd4þCd25� naive

T cells to Cd4þCd25þ regulatory T cells by Tgf-beta induction of

transcription factor Foxp3. J. Exp. Med. 198, 1875–86.

15. Fantini, M. C., Becker, C., Monteleone, G., Pallone, F., Galle, P. R, and

Neurath, M. F. (2004). Cutting edge: Tgf-beta induces a regulatory

phenotype in Cd4þCd25� T cells through Foxp3 induction and

down-regulation of Smad7. J. Immunol. 172, 5149–53.

16. Gonnella, P. A., Chen, Y., Inobe, J., Komagata, Y., Quartulli, M., and

Weiner, H. L. (1998). In situ immune response in gut-associated

lymphoid tissue (Galt) following oral antigen in Tcr-transgenic mice.

J. Immunol. 160, 4708–18.

17. Jang, M. H., Kweon, M. N., Iwatani, K., Yamamoto, M., Terahara, K.,

Sasakawa, C., Suzuki, T., Nochi, T., Yokota, Y., Rennert, P. D., Hiroi, T.,

Tamagawa, H., Iijima, H., Kunisawa, J., Yuki, Y., and Kiyono, H. (2004).

Intestinal villous M cells: an antigen entry site in the mucosal

epithelium. Proc. Natl Acad. Sci. U S A 101, 6110–15.

18. Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G.,

Bonasio, R., Granucci, F., Kraehenbuhl, J. P., and Ricciardi-Castagnoli, P.

(2001). Dendritic cells express tight junction proteins and

penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol.

2, 361–7.

19. Niess, J. H., Brand, S., Gu, X., Landsman, L., Jung, S., McCormick, B. A.,

Vyas, J. M., Boes, M., Ploegh, H. L., Fox, J. G., Littman, D. R., and

Reinecker, H. C. (2005). Cx3cr1-Mediated dendritic cell access to

the intestinal lumen and bacterial clearance. Science 307, 254–8.

20. Huang, F. P., Platt, N., Wykes, M., Major, J. R., Powell, T. J., Jenkins, C. D.,

and MacPherson, G. G. (2000). A discrete subpopulation of dendritic

cells transports apoptotic intestinal epithelial cells to T cell areas of

mesenteric lymph nodes. J. Exp. Med. 191, 435–44.

21. MacPherson, G. G., Jenkins, C. D., Stein, M. J., and Edwards, C. (1995).

Endotoxin-mediated dendritic cell release from the intestine.

Characterization of released dendritic cells and Tnf dependence.

J. Immunol. 154, 1317–22.

22. Roake, J. A., Rao, A. S., Morris, P. J., Larsen, C. P., Hankins, D. F., and

Austyn, J. M. (1995). Dendritic cell loss from nonlymphoid tissues after

systemic administration of lipopolysaccharide, tumor necrosis factor,

and interleukin 1. J. Exp. Med. 181, 2237–47.

�

236

C.

BE

CK

ER

23. Bouma, G. and Strober, W. (2003). The immunological and genetic

basis of inflammatory bowel disease. Nat. Rev. Immunol. 3, 521–33.

24. Guarner, F. and Malagelada, J. R. (2003). Gut flora in health and

disease. Lancet 361, 512–19.

25. Shanahan, F. (2002). Crohn’s disease. Lancet 359, 62–9.

26. Swidsinski, A., Ladhoff, A., Pernthaler, A., Swidsinski, S., Loening-

Baucke, V., Ortner, M., Weber, J., Hoffmann, U., Schreiber, S., Dietel, M.,

and Lochs, H. (2002). Mucosal flora in inflammatory bowel disease.

Gastroenterology 122, 44–54.

27. Linskens, R. K., Huijsdens, X. W., Savelkoul, P. H., Vandenbroucke-Grauls,

C. M., and Meuwissen, S. G. (2001). The bacterial flora in inflammatory

bowel disease: current insights in pathogenesis and the influence of

antibiotics and probiotics. Scand. J. Gastroenterol. Suppl. 29–40.

28. Ogura, Y., Bonen, D. K., Inohara, N., Nicolae, D. L., Chen, F. F.,

Ramos, R., Britton, H., Moran, T., Karaliuskas, R., Duerr, R. H., Achkar,

J. P., Brant, S. R., Bayless, T. M., Kirschner, B. S., Hanauer, S. B.,

Nunez, G., and Cho, J. H. (2001). A frame shift mutation in Nod2

associated with susceptibility to Crohn’s disease. Nature 411, 603–6.

29. Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J.,

Fukase, K., Inamura, S., Kusumoto, S., Hashimoto, M., Foster, S. J.,

Moran, A. P., Fernandez-Luna, J. L., and Nunez, G. (2003).

Host recognition of bacterial muramyl dipeptide mediated

through Nod2. Implications for Crohn’s disease. J. Biol. Chem.

278, 5509–12.

30. Watanabe, T., Kitani, A., Murray, P. J., and Strober, W. (2004). Nod2

is a negative regulator of Toll-like receptor 2-mediated T helper type 1

responses. Nat. Immunol. 5, 800–8.

31. Leach, M. W., Bean, A. G., Mauze, S., Coffman, R. L., and Powrie, F. (1996).

Inflammatory bowel disease in C.B-17 Scid mice reconstituted with

the Cd45rbhigh subset of Cd4þ T cells. Am. J. Pathol. 148, 1503–15.

32. Powrie, F., Leach, M. W., Mauze, S., Caddle, L. B., and Coffman, R. L.

(1993). Phenotypically distinct subsets of Cd4þ T cells induce or protect

from chronic intestinal inflammation in C.B-17 Scid mice. Int. Immunol.

5, 1461–71.

33. Leithauser, F., Trobonjaca, Z., Moller, P., and Reimann, J. (2001).

Clustering of colonic lamina propria Cd4(þ) T cells to subepithelial

dendritic cell aggregates precedes the development of colitis in a murine

adoptive transfer model. Lab. Invest. 81, 1339–49.

34. Malmstrom, V., Shipton, D., Singh, B., Al-Shamkhani, A., Puklavec, M. J.,

Barclay, A. N., and Powrie, F. (2001). Cd134l expression on dendritic cells

�237

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

in the mesenteric lymph nodes drives colitis in T cell-restored Scid mice.

J. Immunol. 166, 6972–81.

35. Krajina, T., Leithauser, F., Moller, P., Trobonjaca, Z., and Reimann, J.

(2003). Colonic lamina propria dendritic cells in mice with Cd4þ T

cell-induced colitis. Eur. J. Immunol. 33, 1073–83.

36. Fuss, I. J., Neurath, M., Boirivant, M., Klein, J. S., de la Motte, C.,

Strong, S. A., Fiocchi, C., and Strober, W. (1996). Disparate Cd4þ lamina

propria (Lp) lymphokine secretion profiles in inflammatory bowel

disease. Crohn’s disease Lp cells manifest increased secretion of

Ifn-gamma, whereas ulcerative colitis Lp cells manifest increased secretion

of Il-5. J. Immunol. 157, 1261–70.

37. Stuber, E., Strober, W., and Neurath, M. (1996). Blocking the

Cd40l–Cd40 interaction in vivo specifically prevents the priming of

T helper 1 cells through the inhibition of interleukin 12 secretion.

J. Exp. Med. 183, 693–8.

38. Monteleone, G., Biancone, L., Marasco, R., Morrone, G., Marasco, O.,

Luzza, F., and Pallone, F. (1997). Interleukin 12 is expressed and actively

released by Crohn’s disease intestinal lamina propria mononuclear cells.

Gastroenterology 112, 1169–78.

39. Neurath, M. F., Fuss, I., Kelsall, B. L., Stuber, E., and Strober, W. (1995).

Antibodies to interleukin 12 abrogate established experimental colitis in

mice. J. Exp. Med. 182, 1281–90.

40. Simpson, S. J., Shah, S., Comiskey, M., de Jong, Y. P., Wang, B.,

Mizoguchi, E., Bhan, A. K., and Terhorst, C. T. (1998). T cell-mediated

pathology in two models of experimental colitis depends predominantly on

the interleukin 12/signal transducer and activator of transcription (Stat)-4

pathway, but is not conditional on interferon gamma expression by T cells.

J. Exp. Med. 187, 1225–34.

41. Davidson, N. J., Hudak, S. A., Lesley, R. E., Menon, S., Leach, M. W., and

Rennick, D. M. (1998). Il-12, but not Ifn-gamma, plays a major role in

sustaining the chronic phase of colitis in Il-10-deficient mice. J. Immunol.

161, 3143–9.

42. Wirtz, S., Finotto, S., Kanzler, S., Lohse, A. W., Blessing, M., Lehr, H. A.,

Galle, P. R., and Neurath, M. F. (1999). Cutting edge: chronic intestinal

inflammation in Stat-4 transgenic mice: characterization of disease and

adoptive transfer by Tnf- plus Ifn-gamma-producing Cd4þ T cells that

respond to bacterial antigens. J. Immunol. 162, 1884–8.

43. Mannon, P. J., Fuss, I. J., Mayer, L., Elson, C. O., Sandborn, W. J.,

Present, D., Dolin, B., Goodman, N., Groden, C., Hornung, R. L.,

Quezado, M., Neurath, M. F., Salfeld, J., Veldman, G. M., Schwertschlag,

�

238

C.

BE

CK

ER

U., Strober, W., and Yang, Z. (2004). Anti-interleukin-12 antibody for active

Crohn’s disease. N. Engl. J. Med. 351, 2069–79.

44. Oppmann, B., Lesley, R., Blom, B., Timans, J. C., Xu, Y., Hunte, B., Vega, F.,

Yu, N., Wang, J., Singh, K., Zonin, F., Vaisberg, E., Churakova, T.,

Liu, M., Gorman, D., Wagner, J., Zurawski, S., Liu, Y., Abrams, J. S., Moore,

K. W., Rennick, D., de Waal-Malefyt, R., Hannum, C., Bazan, J. F., and

Kastelein, R. A. (2000). Novel P19 protein engages Il-12p40 to form a

cytokine, Il-23, with biological activities similar as well as distinct from

Il-12. Immunity 13, 715–25.

45. Cua, D. J., Sherlock, J., Chen, Y., Murphy, C. A., Joyce, B., Seymour, B.,

Lucian, L., To, W., Kwan, S., Churakova, T., Zurawski, S., Wiekowski, M.,

Lira, S. A., Gorman, D., Kastelein, R. A., and Sedgwick, J. D. (2003).

Interleukin-23 rather than interleukin-12 is the critical cytokine for

autoimmune inflammation of the brain. Nature 421, 744–8.

46. Murphy, C. A., Langrish, C. L., Chen, Y., Blumenschein, W.,

McClanahan, T., Kastelein, R. A., Sedgwick, J. D., and Cua, D. J. (2003).

Divergent pro- and antiinflammatory roles for Il-23 and Il-12 in joint

autoimmune inflammation. J. Exp. Med. 198, 1951–7.

47. Wiekowski, M. T., Leach, M. W., Evans, E. W., Sullivan, L., Chen, S. C.,

Vassileva, G., Bazan, J. F., Gorman, D. M., Kastelein, R. A., Narula, S., and

Lira, S. A. (2001). Ubiquitous transgenic expression of the Il-23 subunit P19

induces multiorgan inflammation, runting, infertility, and premature

death. J. Immunol. 166, 7563–70.

48. Stallmach, A., Giese, T., Schmidt, C., Ludwig, B., Mueller-Molaian, I.,

and Meuer, S. C. (2004). Cytokine/chemokine transcript profiles reflect

mucosal inflammation in Crohn’s disease. Int. J. Colorectal Dis. 19, 308–15.

49. Schmidt, C., Giese, T., Ludwig, B., Mueller-Molaian, I., Marth, T.,

Zeuzem, S., Meuer, S. C., and Stallmach, A. (2005). Expression of

interleukin-12-related cytokine transcripts in inflammatory bowel

disease: elevated interleukin-23p19 and interleukin-27p28 in Crohn’s

disease but not in ulcerative colitis. Inflamm. Bowel Dis. 11, 16–23.

50. Fujino, S., Andoh, A., Bamba, S., Ogawa, A., Hata, K., Araki, Y., Bamba, T.,

and Fujiyama, Y. (2003). Increased expression of interleukin 17

in inflammatory bowel disease. Gut 52, 65–70.

51. Nielsen, O. H., Kirman, I., Rudiger, N., Hendel, J., and Vainer, B. (2003).

Upregulation of interleukin-12 and -17 in active inflammatory bowel

disease. Scand. J. Gastroenterol. 38, 180–5.

52. Parham, C., Chirica, M., Timans, J., Vaisberg, E., Travis, M., Cheung, J.,

Pflanz, S., Zhang, R., Singh, K. P., Vega, F., To, W., Wagner, J.,

O’Farrell, A. M., McClanahan, T., Zurawski, S., Hannum, C., Gorman, D.,

�239

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

Rennick, D. M., Kastelein, R. A., de Waal Malefyt, R., and Moore, K. W.

(2002). A receptor for the heterodimeric cytokine Il-23 is composed of

Il-12rbeta1 and a novel cytokine receptor subunit, Il-23r. J. Immunol.

168, 5699–708.

53. Eijnden, S. V., Goriely, S., De Wit, D., Willems, F., and Goldman, M.

(2005). Il-23 up-regulates Il-10 and induces Il-17 synthesis by poly-

clonally activated naive T cells in human. Eur. J. Immunol. 35, 469–75.

54. Aggarwal, S., Ghilardi, N., Xie, M. H., de Sauvage, F. J., and Gurney, A. L.

(2003). Interleukin-23 promotes a distinct Cd4 T cell activation state

characterized by the production of interleukin-17. J. Biol. Chem.

278, 1910–14.

55. Happel, K. I., Zheng, M., Young, E., Quinton, L. J., Lockhart, E.,

Ramsay, A. J., Shellito, J. E., Schurr, J. R., Bagby, G. J., Nelson, S., and

Kolls, J. K. (2003). Cutting edge: roles of Toll-like receptor 4 and Il-23

in Il-17 expression in response to Klebsiella pneumoniae infection.

J. Immunol. 170, 4432–6.

56. Langrish, C. L., Chen, Y., Blumenschein, W. M., Mattson, J., Basham, B.,

Sedgwick, J. D., McClanahan, T., Kastelein, R. A., and Cua, D. J. (2005).

Il-23 drives a pathogenic T cell population that induces autoimmune

inflammation. J. Exp. Med. 201, 233–40.

57. Kolls, J. K. and Linden, A. (2004). Interleukin-17 family members and

inflammation. Immunity 21, 467–76.

58. Fossiez, F., Djossou, O., Chomarat, P., Flores-Romo, L., Ait-Yahia, S.,

Maat, C., Pin, J. J., Garrone, P., Garcia, E., Saeland, S., Blanchard, D.,

Gaillard, C., Das Mahapatra, B., Rouvier, E., Golstein, P., Banchereau, J.,

and Lebecque, S. (1996). T cell interleukin-17 induces stromal cells to

produce proinflammatory and hematopoietic cytokines. J. Exp. Med.

183, 2593–603.

59. Jovanovic, D. V., Di Battista, J. A., Martel-Pelletier, J., Jolicoeur, F. C.,

He, Y., Zhang, M., Mineau, F., and Pelletier, J. P. (1998). Il-17 stimulates

the production and expression of proinflammatory cytokines, Il-beta

and Tnf-alpha, by human macrophages. J. Immunol. 160, 3513–21.

60. Molet, S., Hamid, Q., Davoine, F., Nutku, E., Taha, R., Page, N.,

Olivenstein, R., Elias, J., and Chakir, J. (2001). Il-17 is increased in

asthmatic airways and induces human bronchial fibroblasts to produce

cytokines. J. Allergy Clin. Immunol. 108, 430–8.

61. Antonysamy, M. A., Fanslow, W. C., Fu, F., Li, W., Qian, S., Troutt, A. B.,

and Thomson, A. W. (1999). Evidence for a role of Il-17 in organ allograft

rejection: Il-17 promotes the functional differentiation of dendritic cell

progenitors. J. Immunol. 162, 577–84.

�

240

C.

BE

CK

ER

62. Wu, C. Y., Kirman, J. R., Rotte, M. J., Davey, D. F., Perfetto, S. P., Rhee, E. G.,

Freidag, B. L., Hill, B. J., Douek, D. C., and Seder, R. A. (2002). Distinct

lineages of T(H)1 cells have differential capacities for memory cell

generation in vivo. Nat. Immunol. 3, 852–8.

63. Powrie, F., Leach, M. W., Mauze, S., Menon, S., Caddle, L. B., and

Coffman, R. L. (1994). Inhibition of Th1 responses prevents inflamma-

tory bowel disease in Scid mice reconstituted with Cd45rbhi Cd4þ T cells.

Immunity 1, 553–62.

64. Ito, H. and Fathman, C. G. (1997). Cd45rbhigh Cd4þ T cells from

Ifn-gamma knockout mice do not induce wasting disease. J. Autoimmun.

10, 455–9.

65. Bregenholt, S., Brimnes, J., Nissen, M. H., and Claesson, M. H. (1999).

In vitro activated Cd4þ T cells from interferon-gamma (Ifn-gamma)-

deficient mice induce intestinal inflammation in immunodeficient hosts.

Clin. Exp. Immunol. 118, 228–34.

66. Berg, D. J., Davidson, N., Kuhn, R., Muller, W., Menon, S., Holland, G.,

Thompson-Snipes, L., Leach, M. W., and Rennick, D. (1996). Enterocolitis

and colon cancer in interleukin-10-deficient mice are associated with

aberrant cytokine production and Cd4(þ) Th1-like responses.

J. Clin. Invest. 98, 1010–20.

67. Pflanz, S., Timans, J. C., Cheung, J., Rosales, R., Kanzler, H., Gilbert, J.,

Hibbert, L., Churakova, T., Travis, M., Vaisberg, E., Blumenschein, W. M.,

Mattson, J. D., Wagner, J. L., To, W., Zurawski, S., McClanahan, T. K.,

Gorman, D. M., Bazan, J. F., de Waal Malefyt, R., Rennick, D., and Kastelein,

R. A. (2002). Il-27, a heterodimeric cytokine composed of Ebi3 and P28

protein, induces proliferation of naive Cd4(þ) Tcells. Immunity 16, 779–90.

68. Lucas, S., Ghilardi, N., Li, J., and De Sauvage, F. J. (2003). Il-27 regulates

Il-12 responsiveness of naive Cd4þ T cells through Stat1-dependent

and -independent mechanisms. Proc. Natl Acad. Sci. U S A 100, 15047–52.

69. Takeda, A., Hamano, S., Yamanaka, A., Hanada, T., Ishibashi, T.,

Mak, T. W., Yoshimura, A., and Yoshida, H. (2003). Cutting edge: role of

Il-27/Wsx-1 signaling for induction of T-Bet through activation of Stat1

during initial Th1 commitment. J. Immunol. 170, 4886–90.

70. Nieuwenhuis, E. E., Neurath, M. F., Corazza, N., Iijima, H., Trgovcich, J.,

Wirtz, S., Glickman, J., Bailey, D., Yoshida, M., Galle, P. R.,

Kronenberg, M., Birkenbach, M., and Blumberg, R. S. (2002). Disruption of

T helper 2-immune responses in Epstein–Barr virus-induced gene

3-deficient mice. Proc. Natl Acad. Sci. U S A 99, 16951–6.

71. Wang, J. and Fu, Y. X. (2005). Tumor necrosis factor family members

and inflammatory bowel disease. Immunol. Rev. 204, 144–55.

�241

DE

ND

RIT

ICC

EL

LS

INT

HE

GU

TA

ND

TH

EIR

PO

SS

IBL

ER

OL

EIN

DIS

EA

SE

72. Atreya, R., Mudter, J., Finotto, S., Mullberg, J., Jostock, T., Wirtz, S.,

Schutz, M., Bartsch, B., Holtmann, M., Becker, C., Strand, D., Czaja, J.,

Schlaak, J. F., Lehr, H. A., Autschbach, F., Schurmann, G.,

Nishimoto, N., Yoshizaki, K., Ito, H., Kishimoto, T., Galle, P. R.,

Rose-John, S., and Neurath, M. F. (2000). Blockade of interleukin 6

trans signaling suppresses T-cell resistance against apoptosis in

chronic intestinal inflammation: evidence in Crohn’s disease and

experimental colitis in vivo. Nat. Med. 6, 583–8.

73. Strober, W., Fuss, I. J., and Blumberg, R. S. (2002). The immunology

of mucosal models of inflammation. Annu. Rev. Immunol. 20, 495–549.

74. Yamanaka, T., Helgeland, L., Farstad, I. N., Fukushima, H., Midtvedt, T.,

and Brandtzaeg, P. (2003). Microbial colonization drives lymphocyte

accumulation and differentiation in the follicle-associated epithelium

of Peyer’s patches. J. Immunol. 170, 816–22.

75. Onderdonk, A. B., Richardson, J. A., Hammer, R. E., and Taurog, J. D.

(1998). Correlation of cecal microflora of Hla-B27 transgenic rats

with inflammatory bowel disease. Infect. Immun. 66, 6022–3.

76. Rath, H. C., Ikeda, J. S., Linde, H. J., Scholmerich, J., Wilson, K. H.,

and Sartor, R. B. (1999). Varying cecal bacterial loads influences

colitis and gastritis in Hla-B27 transgenic rats.Gastroenterology 116, 310–19.

77. Mizoguchi, A., Mizoguchi, E., Chiba, C., and Bhan, A. K. (1996). Role

of appendix in the development of inflammatory bowel disease in Tcr-alpha

mutant mice. J. Exp. Med. 184, 707–15.

78. May, E., Lambert, C., Holtmeier, W., Hennemann, A., Zeitz, M.,

and Duchmann, R. (2002). Regional variation of the alphabeta T cell

repertoire in the colon of healthy individuals and patients with Crohn’s

disease. Hum. Immunol. 63, 467–80.

79. Kuckelkorn, U., Ruppert, T., Strehl, B., Jungblut, P. R., Zimny-Arndt, U.,

Lamer, S., Prinz, I., Drung, I., Kloetzel, P. M., Kaufmann, S. H., and

Steinhoff, U. (2002). Link between organ-specific antigen processing by 20s

proteasomes and Cd8(þ) T cell-mediated autoimmunity. J. Exp. Med.

195, 983–90.

80. Becker, C., Wirtz, S., Blessing, M., Pirhonen, J., Strand, D., Bechthold, O.,

Frick, J., Galle, P. R., Autenrieth, I., and Neurath, M. F. (2003). Constitutive

P40 promoter activation and Il-23 production in the terminal ileum

mediated by dendritic cells. J. Clin. Invest. 112, 693–706.

�

242

C.

BE

CK

ER

Index

Adaptive immunity 194–195

Adenylate cyclase toxin (CyaA) 200

Antigen uptake 225–226

b2-microglobulin 53

Bacillusanthracis 200, 205

subtilis 86Bacterial receptors

on antigen presenting cells 173–175

combination of, 175–176

cross-talk between pathogen

receptors

Bacterial toxins 200, 201

Bacterial uptake 82, 83, 225–226

Bacteroides thetaiotamicron 86

Birc1e 143

Bordetella pertussis 198, 202, 207, 211

Borrelia burgdorferi 197, 198

Brucella abortusantigen presentation 62

Calnexin 52

Calreticulin 52

Candida albicans 39, 182, 183

CARD domain 37–38

Caspase-1 107

CCL20 86

CCR2 104

CCR9 87

CD45RBhi 231

CD103 87

Cholera toxin (CT) 197, 200, 205

Colitis, bacteria-induced 204

Complement pathway 194, 195

Costimulatory molecules 205

Coxiella burnetii 206CpG-DNA 28, 35–36, 107

Crohn’s, disease 37, 90, 223, 228, 232

Cross-presentation 9, 160

CX3CL1 82

CX3CR1 226

C-type lectins (CLR) 174

in immune responses 179

cross-talk with TLR 179–183

Cyclic AMP (cAMP) 205

DC-SIGN 175, 179, 183

DEC-205 175, 179

Dectin-1 175, 182

Defective ribosomal products (DRIPS)

52

Dendritic cells aggresome-like

structures (DALIS) 53

Dendritic cells

as bystander antigen presenting cells

164–165

B220þLy6cþ 13, 84

CD4þ 10

CD8þ 9–10, 84

CD8� 10–11

CD11bþ 11–12, 84, 104

Dendritic cells (cont.)

CD83þCD123þ 85

classification 3

conventional 3–7

cross-presentation 9, 160

cross-talk with epithelial cells 88–91

de-novo formed 6

dermal 12, 13

differentiation 14–17

in vivo depletion of 109

interstitial 12, 13

location 3–6

lymphoid organ, restricted 3–6, 8–12

lymphoid 15

ly49Qþ 13

ly49Qþ 13

maturation in response to bacteria

204–206

migratory 12–13

monocyte-derived 7

mucosal 86, 224

myeloid 15

non-lymphoid organ, restricted 6

Langerhans cells 6, 12

plasmacytoid 7, 13–14

resistance to NK mediated cell death

127–128

TIP-DCs 104–106

tolerogenic 224–225

Di-acyl lipoprotein 27

ERAD 60

Escherichia coli 56, 182

heat labile toxin (LT) 197, 200, 205

presentation of antigen 56

Fc receptor, g 163

Fc receptor, neonatal 83

Filamentous hemagglutinin (FHA) 199,

202

Flagellin 28, 107, 144

Flt3L 16

Follicle associated epithelium 82

Foxp3 207, 225

GM-CSF 16

Gut associated lymphoid tissue 82, 223

Helicobacterhepaticus 204, 210

pylori 181, 197, 204, 211

Herpes simplex 13

Immunoactivation 182–183

and TLR 176–177

cross-talk between CLR and TLR

179–182

T cell suppression 195–198, 209–211

Immunoglobulin A 11, 83, 87, 225

Immunoglobulin G 83, 195

Immunoproeasome 53

Indirect antigen presentation 164

Inflammatory bowel disease 210, 227

Innate immunity 193–194

Interferon, type I 13, 14, 36–37, 90, 147

Interferon, type II 102, 145, 146, 195,

231

Interleukin-4 145

Interleukin-6 108

Interleukin-10 11, 87, 108, 145, 184, 225

induction of 201–204

Interleukin-12 10–13, 88, 108, 144, 184

suppression of 199–201, 205

Interleukin-17 194, 230

Interleukin-23 230

Interleukin-27 232

Internalin A 101

Intestinal mucosa, anatomy 81–2

Invariant chain 63

IRAK 30

IRF-2 17

IRF-3 33

IRF-4 17

IRF-7 33

IRF-8 17

ITAM motif 175, 182

ITIM motif 175

KIR 120, 121

Klebsiella 183

Lactobacillus 88, 182, 201, 202

reuteri 201casei 201

Lamina propria 82

�

244

IND

EX

Legionella pneumophila 141

adaptive immune response 145–146

antigen presentation 62, 149–150

Dot/Icm system 142

growth in dendritic cells 148–149

growth in macrophages 146–148

innate immune response 144

Th1/Th2 balance 145

vacuole survival 70, 142–143

Leishmania major 8, 10, 11, 13, 181

Lipopolysaccharide (LPS) 27, 107

Lipotheicoic acid (LTA) 107

Listeria 32, 53, 201

innate immune response to 102–103

interaction with DCs 106–109

monocytogenes 32, 65, 67, 203

myeloid cell involvement 103–104

pathogenesis 101–102

presentation of antigens 72

Listeriolysin 101

Lox1 184

M cells 82

Mannose receptor (MR) 175, 179, 183

MAP kinase 31

MHC I presentation 52–63

apoptosis and cross-presentation

70–72, 164–165

cross-presentation 51, 58, 60

cytosolic pathway 52–56

‘‘alternative pathway’’ 56

‘‘classical pathway’’ 58

ER-phagosome fusion 60

interference by bacteria 67–70

phagosomal pathway 56–58

MHC I trafficking 57

MHC II presentation 63–67

autophagy 66

developmental regulation 64

endo-lysosomal pathway 63–65

interference by bacteria 67–70

of cytocol-derived antigens 65–67

Mycobacterium 58, 199, 202

bovis BCG 56, 69, 125, 182, 203

leprae 197tuberculosis 39, 68, 176, 181, 182,

202–203

vaccae 209MyD88-dependent, pathway 30–32, 106,

107

NADPH oxidase 99

Natural killer cells 102, 119

adaptive immunity 120

cross-talk with DCs mediated by

bacteria 124–128

editing of DCs 126–127

interaction with DC during infection

128–130

NF-kB 31, 86

Nitric oxide synthase (NOS) 147

Inducible 99, 105, 163

NOD-LRR 143

NOD-1 37, 175

NOD-2 37, 85, 175, 227

Opsonization 164

OspA 197

Outer membrane protein A (ompA) 183

Ox40L 228

Pasteurella multocida toxin (PMT) 201

Pathogens, intracellular recognition

36–38

disturbance of TLR signaling 38–39

Peptidoglycan 37, 107

Phagosome

inhibition of phagolysosomal fusion

68–69

survival of virulent bacteria 69–70

PhoP/PhoQ 162

Porphyromonas gingivalis 177Probiotics 201

Proteasome 52

PTX3 184

Reactive oxygen species (ROS) 184

Reovirus 12

RNA,

double-stranded 28, 36

single stranded 28, 36

Salmonella 56, 86, 182

as anticancer agent 165

�245

IND

EX

Salmonella (cont.)

immunosuppression 196

LPS from 177

Pathogenicity Island-2 (SPI2) 163

presentation of antigens 56, 58, 71,

161

processing and presentation in

macrophages 162

processing in DCs 161–162

survival in DCs 69, 160–161

uptake in the gut 226

Schistosoma 177

mansoni 181, 184

Shigella 53

flexneri 67SIGNR1 182

SREC-1 184

Streptococcus 67gordonii 58pneumoniae 182pyogenes 209

TAP transporters 52

T cell anergy or suppression 195–198

Th1 cells 194

Th2 cells 194

Th IL17 230

Th1/Th2 axes 88–90, 130, 145, 181, 197

in Crohn’s disease 228

Thymic stromal lymphopoietin (TSLP)

88

TIR, domain 28

Tight junctions 81, 226

Toll-like receptors (TLRs) 14, 27–28, 85,

173

cross-talk with CLR 179–183

in immune evasion 176–177

signaling in conventional DCs 28–34

signaling in plasmacytoid DCs 34–36

Toxoplasma gondii 10, 28

TRAF-6 17

T regulatory cells 90, 194

bacteria-induced 207–209

in bacterial infections 209–211

in inflammatory bowel disease 210

natural and inducible 206–207

Tri-acyl lipoprotein 28

TRIF-dependent, pathway 32–34

Tumor growth factor (TGF)-b 201–204,

225

Tumor necrosis factor (TNF)-a 102, 105,

199–201, 205

Ulcerative colitis 223, 232

Yersinia 177, 202, 226

enterocolitica 201, 202, 206

pseudotuberculosis 197

�

246

IND

EX